Method for selective inactivation of viral replication

ABSTRACT

Method for screening for an antiviral agent, by determining whether a potential agent interacts with a virus or cellular component which allows or prevents preferential translation of a virus RNA compared to a host RNA under virus infection conditions; and determining whether any interaction of the agent with the component reduces the level of translation of an RNA of the virus.

RELATED APPLICATION

[0001] This application is a continuation-in-part of Miles et al., U.S.patent application Ser. No. 08/042,024, filed Apr. 2, 1993, entitled“Method for Selective Inactivation of Viral Replication,” the whole ofwhich (including drawings, if any) is hereby incorporated by reference.

[0002] This invention relates to methods for screening for agents usefulfor treatment of viral infection, the novel agents identified using suchscreening methods, and their use as antiviral agents.

BACKGROUND OF THE INVENTION

[0003] A variety of agents are presently used to combat viral infection.These agents include interferon, which is a naturally-occurring proteinhaving some efficacy in combat of certain selected viral diseases. Inaddition, agents such as AZT are used in the combat of animmunodeficiency disease, referred to commonly as AIDS, caused by thevirus HIV-1.

[0004] Drug and Market Development, Vol 3. No. 9, pp. 174-180 (Feb. 15,1993), describes antiviral drug development. It states:

[0005] The difficulties encountered in drug treatment of most infectionspale when compared to viral infections. For example, it is at leasttheoretically (and often in practice) possible to attack a bacteriumwithout harming the host. Unlike bacteria however, viruses replicateinside cells and utilize cellular machinery of the host for replication.As a result, development of antiviral therapeutics often represents acompromise between preferable killing, or at least arresting replicationof, the virus, and not harming the host, or at worst, doing only minimaldamage which can be justified by the potential gain.

[0006] It states that viral specific events can be targeted including:

[0007] * Virus attachment to cell membranes and penetration in cells;

[0008] * Virus uncoating;

[0009] * Virus nucleic acid synthesis;

[0010] * Viral protein synthesis and maturation; and

[0011] * Assembly and release of infectious particles.

[0012] Specifically with regard to viral protein synthesis the authorsstate:

[0013] In contrast to nucleic acid synthesis, viral protein synthesisutilizes host ribosomes (ribosomes are cell structures essential fortranslation of mRNA into protein) and mostly host-derived supplementaryfactors. As a result, protein synthesis inhibitors, in general, are aslikely to exhibit host toxicity as they are to exert antiviral effects.Antisense oligonucleotides, however, may be of value in specificallyinhibiting viral protein synthesis. Briefly, antisense oligonucleotidesare short DNA fragments that are complementary to mRNA (sense strands)and can prevent mRNA-directed protein synthesis by binding to mRNA. RNAmolecules have also been constructed to contain sequences complementaryto those of sense DNA strands (and their corresponding mRNA). Althoughantisense constructs have been shown to inhibit viral protein synthesisin vitro, their effectiveness in vivo has not yet been conclusivelydemonstrated. Among others, current challenges for oligonucleotidetherapeutics include delivery to virus-infected cells, the stability ofsuch molecules in vivo and distribution throughout the body.

[0014] Ribosome inactivators represent another approach for viralprotein synthesis inhibition. GLQ223 (Genelabs; Redwood City, Calif.) isa ribosome inactivator undergoing clinical testing (GLQ223 is a purifiedpreparation of trichosanthin (cucumber plant derivative)). A ribosomeinactivator would interfere with cellular translation machinery,effectively preventing generation of new viral proteins.

[0015] Sonenburg, 2 The New Biologist 402, 1990 describes virus hostinteractions at the level of initiation of translation and states thattwo initiation factors eIF-2 and eIF-4F play significant roles in anumber of virus host interactions. He states “[a]n understanding of themechanisms responsible for these virus-host interactions is of greatsignifigance for future therapeutic approaches to viral disease.”

SUMMARY OF THE INVENTION

[0016] The present invention relates to methods for screening for agentswhich are effective in inhibiting the translational system used by avirus during infection of a host cell. The screening method utilizes aprotocol in which potentially useful agents are brought into contactwith appropriate macromolecular sequences, e.g., viral nucleic acidsequences or relevant protein sequences, in order to determine whetherthose agents can specifically inhibit use of those sequences. Virusesuse a variety of methods for taking over a host translational system,and it is these methods that can be specifically targeted by methods ofthe present invention. Once isolated, the viral specific agents can beformulated in therapeutic products (or even prophylactic products) inpharmaceutically acceptable formulations, and used for specifictreatment of viral disease with little or no effect on uninfected virushost cells.

[0017] Specifically, in one aspect, applicant provides a screeningmethod in which a target virus nucleic acid sequence or domainresponsible for preferential translation of viral RNA over host RNA isused in a selection protocol. While several specific examples of suchviral nucleic acid sequences or domains are provided below in the formof IRES elements, 5′-untranslated regions containing specific viralsequences, and upstream open-reading frames containing such sequences,these are used only to exemplify a general method by which other virusnucleic acid sequences can be used in such protocols. Use of any one ofthese virus nucleic acid sequences within a cell translation systemprovides a means by which anti-viral agents can be discovered.

[0018] Applicant notes that the claimed method does not includetargeting of agents to viral sequences involved in frame shifting (whichis not a target nucleic acid that is preferentially translated asdefined herein), such as described by Dinman and Wickner, 66 J. Virol.3669, 1992; Jacke et al., 331 Nature 280, 1988; Wilson et al., 55 Cell1159, 1988; Inglis and Brierly, WO 90/14422; and Goodchild and Zamecnik,WO 87/07300.

[0019] Any agent which binds to such viral nucleic acid and/or whichcauses a significant reduction in translation of viral message ispotentially useful in the present invention. Such agents can be screenedto ensure that they are specific to viral translation systems and haveno effect on uninfected host cell translation systems such that theagent can be used in a therapeutic or prophylactic manner. If suchagents have some effect on host cell systems they may still be useful intherapeutic treatment, particularly in those diseases which are lifethreatening, such as HIV-1 infection.

[0020] Such agents may interact either directly with the target viralnucleic acid, for example, by hybridization with the nucleic acid, e.g.,antisense RNA or DNA, or may bind or interact with other components ofthe viral translation system (i.e., those host and/or viral componentswhether nucleic acid and/or protein which allow translation of viralmRNA to occur in vivo), such as proteins used by the virus to promotetranslation of its RNA, rather than host RNA involved in that system,e.g., antibodies. Additionally, agents may include any nucleic acidmolecule which binds to viral or cellular components which otherwisewould partake in preferential viral nucleic acid translation, but uponbinding said nucleic acid molecule become unable to be preferentiallytranslated. However, while antisense nucleic acid and antibodies mayexemplify aspects of the present invention, applicant is particularlyconcerned with identification of agents of low molecular weight (lessthan 10,000, preferably less than 5,000, and most preferably less than1,000), which can be more readily formulated as useful antiviral agents.Thus, in a preferred embodiment, the invention features such lowmolecular weight agents, and not antisense molecules or antibodies.

[0021] Thus, in a first aspect the invention features a method forscreening for an antiviral agent. The method includes providing a targetviral translation nucleic acid sequence which allows preferentialtranslation of a viral RNA compared to a host RNA under virus infectionconditions. The method may involve a simple assay to detect binding ofan agent to this nucleic acid. Preferably, however, the target viraltranslation nucleic acid sequence is translationally linked to RNAencoding a reporter polypeptide. The method then further includescontacting the target viral translation nucleic acid sequence with apotential antiviral agent under conditions which allow synthesis of thereporter polypeptide in the absence of the agent. The method finallyincludes determining whether the agent reduces the level of translationof the reporter polypeptide. Any agent which does reduce this level ispotentially a useful antiviral agent.

[0022] Specifically, the method involves determining whether a potentialagent interacts with a virus or cellular component which allows orprevents preferential translation of a virus RNA compared to a host RNAunder virus infection conditions; and determining whether anyinteraction of the agent with the component reduces the level oftranslation of a RNA of the virus.

[0023] By “screening” is preferably meant a process in which a largenumber of potentially useful agents are processed in the method of thisinvention. It is generally a process distinct from a single experimentin which a single agent is studied in detail to determine its method ofaction.

[0024] By target viral translation nucleic acid sequence is meant anynucleic acid which allows preferential translation of translationallyassociated RNA under viral infection conditions. Such nucleic acid isexemplified by IRES elements which allow cap-independent translation ofassociated ribonucleic acid, and 5′ untranslated regions of influenzavirus RNA which allow preferential cap-dependant translation ofassociated RNA.

[0025] By preferential translation is meant that the RNA is translatedat a higher rate or with higher yield of protein than host cell RNAunder virus-infection conditions. In addition, the host cell RNA may betranslated at a slower rate or with lower protein yield than innon-infected conditions. Such preferential translation on be readilydetected as described below. In the case of most viruses, preferentialexpression of viral proteins means that synthesis of viral proteinsrepresents at least 50% of total de novo protein synthesis, as may bedetected, for example, by pulse-labeling experiments in viral-infectedcells. In such cases, viral proteins may usually be distinguished asmajor bands when labeled proteins are separated by gel electrophoresis.In the case of retroviruses, preferential expression of viral proteinsmeans that the level of viral proteins synthesized increasesdisproportionately beyond the level of viral RNA synthesized (Cullen,Cell 46: 973, 1986) Such a disproportionate increase can be detected byquantitating levels of viral RNA and protein synthesis in infected cellsby, for example, Northern blotting and nuclease protection assays forRNA synthesis and immunoprecipitations and gel electrophoresis forlabeled proteins.

[0026] By virus infection conditions is simply meant conditions within ahost cell after infection with the target virus such that the viraltranslation system is operative. Such a viral translation system willusually include host cell proteins, nucleic acids and other components.

[0027] By reporter polypeptide is simply meant a peptide which isreadily detectable, either by providing a calorimetric signal undercertain environmental conditions or some other signal well known tothose of ordinary skill in the art, as described below.

[0028] In preferred embodiments, the component is a protein or a nucleicacid; the component is virus encoded or host cell encoded; the componentis a macromolecule selected from an RNA sequence domain, a DNA sequencedomain, an initiation factor, and elongation factor, a terminationfactor, a transcription factor, a ribosomal protein, a glycosylase, adeglycosylase, a prenylating and deprenylating enzyme, a transferase, apolymerase, a synthetase, an ADP ribosylating enzyme, an ADP ribosylase,a kinase, a lipase, a myristylating or demyristylating enzyme, aphosphorylase, a protease, a rRNA, a tRNA, a ribonuclease, and adeoxyribonuclease; the viral translation signal nucleic acid sequence isselected from the group consisting of IRES elements, 5′ or 3′untranslated regions, and upstream open reading frames, or any otherviral target translation nucleic acid that affords preferentialtranslation of viral mRNA over host cell mRNA when the host cells areinfected by the virus; and the virus from which that signal is selectedis chosen from the picornavirus family, Hepatitis viruses A, B,. and C,influenza virus, HIV, Herpes virus, and cytomegalovirus.

[0029] In other preferred embodiments, the sequence domain istranslationally linked to RNA encoding a reporter polypeptide, and thesecond determining step includes determining whether the agent altersthe level of translation of the reporter polypeptide; the component is aprotein or a polypeptide, and the determining steps include providingthe component in a translation mixture with RNA encoding a reporterpolypeptide, and determining whether the agent alters expression of thereporter polypeptide in the mix.

[0030] In more preferred embodiments, the method further includesdetermining whether an agent active in the above method has little or noeffect on the translational machinery of an uninfected viral host cell,and further determining whether the agent is active under in vivoconditions. Such agents are then formulated in a pharmaceuticallyacceptable buffer.

[0031] By pharmaceutically acceptable buffer is meant any buffer whichcan be used in a pharmaceutical composition prepared for storage andsubsequent administration, which comprise a pharmaceutically effectiveamount of an agent as described herein in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and evenflavoring agents may be provided in the pharmaceutical composition. Forexample, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

[0032] In a second aspect, the invention features a method for treatinga subject infected with a virus having a viral translation signalnucleic acid sequence, by administering to that subject atherapeutically effective amount of an antiviral agent able toselectively block translation of viral RNA naturally linked to the viraltranslation signal nucleic acid sequence.

[0033] By “therapeutically effective amount” is meant an amount thatrelieves (to some extent) one or more symptoms of the disease orcondition in the patient. Additionally, by “therapeutically effectiveamount” is meant an amount that returns to normal, either partially orcompletely, physiological or biochemical parameters associated with orcausative of a viral disease. Generally, it is an amount between about 1nmole and 1 μmole of the molecule, dependent on its EC₅₀ and on the age,size, and disease associated with the patient.

[0034] In a third related aspect, the invention features novel antiviralagents discovered by the methods described above. It also includes novelpharmaceutical compositions which include antiviral agents, discoveredas described above, and formulated in pharmaceutically acceptableformulations.

[0035] In a fourth aspect, the invention features the use of nucleicacid constructs containing isolated viral nucleic acid translationallylinked to a reporter-encoding sequence to discover antiviral agents, andkits for use of these constructs in antiviral agent screening methods.

[0036] In a fifth aspect, the present invention features a screeningmethod for antiviral agents active at modulating the activity of other,non-nucleic acid, macromolecules involved in the viral mRNA translationsystem. For example, the method for screening agents includesidentifying those effective at inhibiting macromolecules that interferewith the activity of such macromolecules, e.g., agents which allow thep68 kinase in a cell to exhibit its activity. The invention alsofeatures a method of employing such agents for inhibiting replication ofvirus in eukaryotic host cells.

[0037] Thus, the invention includes a method of inhibiting viralreplication in a host eukaryotic cell, e.g., where the virus produces aviral inhibitor which interferes with the activation of a host-cellinterferon-induced, double-stranded RNA-activated protein kinase. Themethod includes administering to the cells, an agent able to block theeffect of the viral inhibitor in interfering with the activation of theprotein kinase.

[0038] In a related aspect, the invention features a virus whichproduces a viral inhibitor able to block binding of double-stranded RNAto the protein kinase, and the agent administered is one able to blockthe binding of the viral inhibitor to the protein kinase.

[0039] The agent may be selected, for example, by forming a mixturecomposed of protein kinase, the viral inhibitor, and the agent,incubating the components of the binding mixture under conditionseffective to bind the protein kinase to the viral inhibitor, in theabsence of the agent, and examining the mixture for the presence ofbinding of the protein kinase to the viral inhibitor, to determinewhether the presence of the test agent has inhibited binding the proteinkinase to the viral inhibitor.

[0040] Alternatively, the agent may be selected by forming a mixturecomposed of protein kinase, the viral inhibitor, and the agent,incubating the components of the mixture under conditions effective toautophosphorylate the protein kinase in the absence of the viralinhibitor, examining the mixture for the presence of protein kinaseactivity, and selecting the agent if it is able to prevent inhibition ofprotein kinase activity.

[0041] In specific examples, where the virus is an adenovirus, and theviral inhibitor is a VAI RNA molecule (also known as VA 1 and VARNA₁);the virus is human immunodeficiency virus (HIV), and the viral inhibitoris a TAR region of the HIV genome; and the virus is an Epstein-Barrvirus, and the viral inhibitor is an EBER-1 RNA.

[0042] In another related aspect, the viral inhibitor is effective toactivate a host-cell p58 protein which is able, in activated form, toblock the activation of the protein kinase, or to block the activity ofalready activated protein kinase, and the agent is one which blocks theinteraction of the viral inhibitor through p58 protein on the kinase.The agent may be selected, for example, by forming a mixture composed ofprotein kinase, the p58 protein (an active form), and the agent, andthen incubating the components of the mixture under conditions effectiveto autophosphorylate the protein kinase, when the p58 protein is absent,examining the mixture for the presence of protein kinase activity, andselecting the agent if it is able to reduce inhibition of protein kinaseactivity, when p58 is present.

[0043] In another related aspect, the invention includes a method forscreening agents effective to inhibit viral replication in a hosteukaryotic cell, where the virus is one able to produce a viralinhibitor which interferes with the activation of the host-cellinterferon-induced, double-stranded RNA-activated protein kinase. Themethod includes incubating a mixture containing the protein kinase, theviral inhibitor, and the agent to be tested, under conditions effectiveto cause viral inhibitor interference with the activation of the proteinkinase, and examining in mixture for such interference.

[0044] The method of this invention can also be used for screening anagent effective to inhibit replication in a host cell of a virus whichproduces a viral inhibitor capable of binding to the protein kinase, toinhibit binding of double-stranded RNA to the protein kinase. In thismethod, the mixture is incubated under conditions effective to bind theviral inhibitor to the protein kinase, and the mixture is examined forbinding of the viral inhibitor to the protein kinase. The incubating maybe carried out, for example, in solution phase, and the examining stepincludes passing the mixture through a filter which retains the viralinhibitor only when the inhibitor is bound to the protein kinase.Alternatively, the protein kinase may be bound to a solid support, theviral inhibitor labeled with a reporter, and the examining stepperformed by measuring the amount of reporter bound to the solidsupport. In addition, the incubating may be carried out under conditionsin which the protein kinase is autophosphorylated, in the absence ofbinding to the viral inhibitor, and the examining step performed bydetermining the extent of phosphorylation of the p68 kinase.

[0045] In another related aspect, the method of this invention is usedfor screening agents effective in blocking viral replication of a viruswhich produces an viral inhibitor effective to activate a p58 host-cellprotein which in activated form is effective to blockautophosphorylation of the protein kinase or to block activity of thephosphorylated kinase. Here the mixture formed includes the p58host-cell protein, the incubating step is carried out under conditionsin which the protein kinase would be autophosphorylated in the absenceof p58, and the mixture is examined for reduction of inhibition ofprotein kinase activity.

[0046] In still another aspect, the protein kinase and viral inhibitorare expressed in a yeast cell which is constructed to increase theexpression of a marker protein in the presence of activated proteinkinase, and the yeast cells are examined for increased expression of themarker protein. This aspect concerns use of a yeast cell in screeningagents effective to inhibit viral replication in a host eukaryotic cell,where the virus is able to produce a viral inhibitor which interfereswith the activation of the host-cell interferon-induced, double-strandedRNA-activated protein kinase. The cell includes (a) an expressible geneencoding a mammalian interferon-induced, double-stranded RNA-activatedprotein kinase, (b) a reporter gene whose expression in increased byactivation of the protein kinase, and (c) a viral gene for producing aviral inhibitor able to block activation of the protein kinase.

[0047] In yet other preferred embodiments, the method of this inventionincludes forming a protein translation mixture which includes (i) aviral mRNA construct, the mRNA construct comprising (a) an internalribosome entry site (IRES) region and downstream of the IRES region, afirst reporter protein coding region, (ii) ribosomes, and (iii) an agentto be tested, incubating the components of the translation mixture underconditions effective to produce from the first reporter protein codingregion a reporter protein, and examining the mixture for the presence ofreporter protein produced by such translation mixture, and the agent isa useful anti virus agent if the reporter protein produced in thepresence of the test agent is less than an amount of reporter proteinproduced in the absence of the test agent.

[0048] Preferably, the IRES region is derived from a picornavirus IRESregion sequence; the IRES sequence is selected from the group consistingof an enterovirus, rhinovirus, cardiovirus, and aphthovirus IRESsequence; the IRES region is selected from the group consisting of anhepatitis A virus IRES sequence, an hepatitis B virus sequence and anhepatitis C virus IRES sequence; the protein translation mixture is acell-free extract; the 5′-end of the viral mRNA construct. includes aeukaryotic mRNA 5′-terminal cap and untranslated region (UTR) anddownstream of the cap and UTR region, a second reporter protein; and thetranslation mixture is contained in a cell.

[0049] In another example, the method includes forming a binding mixturecomprising a cellular or viral translation initiation protein, an IRESelement ribonucleotide sequence, and an agent to be tested, incubatingthe components of the binding mixture under conditions effective to bindthe initiation protein to the IRES element, and examining the mixturefor the presence of binding of the initiation protein to the IRESelement. The agent is a useful antivirus agent if the extent of bindingof the initiation protein to the IRES element is less than that observedin the absence of the agent.

[0050] Preferably, the cellular or viral translation initiation proteinis selected from the group consisting of p52 and p57; the cellular orviral translation initiation protein is bound to a solid support, theIRES element is labeled with a reporter, and the examining includesmeasuring the amount of reporter bound to the solid support; the IRESelement RNA is bound to a solid support, the cellular or viraltranslation initiation protein is labeled with a reporter, and theexamining includes measuring the amount of reporter bound to the solidsupport; a terminal region of the IRES element is bound to acomplementary DNA sequence, and the DNA sequence is linked to the solidsupport; and the method further includes the step, after the incubatingstep, of adding to the incubation mixture an RNAase capable of cleavingfree RNA but not protein bound RNA, and the binding of the initiationprotein to the IRES element is detected by the presence in the mixtureof uncleaved IRES element RNA.

[0051] In one example, the examining includes subjecting the mixture toa gel-shift electrophoresis assay.

[0052] In still other preferred embodiments, the incubating is carriedout in solution phase, and the examining includes passing the mixturethrough a filter which retains the IRES element only when the element isbound to the cellular or viral translation initiation protein.

[0053] In a related aspect, the agent is effective to inhibit viralreplication in a host eukaryotic cell, where the virus produces aninhibitor which interferes with the activation or activity of thehost-cell interferon-induced, double-stranded RNA-activated proteinkinase, and the screening method includes incubating a mixturecontaining the protein kinase, the inhibitor, and the agent to be testedunder conditions effective to cause inhibitor interference with theactivation or activity of the protein kinase, and examining the mixturefor such interference; or the agent is effective to inhibit viralreplication in a host eukaryotic cell, where the host cell produces aninhibitor which interferes with the activation of the host-cellinterferon-induced, double-stranded RNA-activated protein kinase, andthe method includes incubating a mixture containing the protein kinase,the inhibitor, and the agent to be tested under conditions effective tocause inhibitor interference with the activation of the protein kinase,and examining the mixture for such interference.

[0054] Preferably, the method is for use in screening an agent effectiveto inhibit replication in a host cell of a virus which produces aninhibitor able to bind to the protein kinase, to interfere with theactivation of the protein kinase by double-stranded RNA, and theincubating includes incubating the protein kinase, viral inhibitor, andagent under conditions effective to bind the inhibitor to the proteinkinase, and the examining includes examining the protein kinase forbound inhibitor; or the incubating is carried out in solution phase, andthe examining includes passing the protein kinase, viral inhibitor, andtest agent through a filter which retains the inhibitor only when theinhibitor is bound to the protein kinase; or the protein kinase is boundto a solid support, the inhibitor is labeled with a reporter, and theexamining includes measuring the amount of reporter bound to the solidsupport; or the incubating is carried out under conditions in which theprotein kinase is autophosphorylated, in the absence of binding to theviral inhibitor, and the examining includes determining the extent ofphosphorylation of the p68 kinase; or the method is for use in screeningagents effective in blocking viral replication of a virus which producesan inhibitor effective to activate a p58 host-cell protein which inactivated form is effective to block activity or activation of theprotein kinase, and the mixture formed includes the p58 host-cellprotein, the incubating is carried out under conditions in which theprotein kinase is activated in the absence of p58, and the examiningincludes examining the mixture for inhibition of protein kinaseactivity.

[0055] In a preferred embodiment, the protein kinase and inhibitor areexpressed in a yeast cell which is constructed to increase theexpression of a reporter protein in the presence of activated proteinkinase, and the examining includes examining the yeast cells forincreased expression of the reporter protein; and the reporter proteinis fused GCN4/β-gal protein.

[0056] In another aspect, the invention features a yeast cell for use inscreening agents effective to inhibit viral replication in a hosteukaryotic cell, where the virus produces a viral inhibitor whichinterferes with the activation of the host-cell interferon-induced,double-stranded RNA-activated protein kinase. The cell includes (a) anexpressed gene encoding a mammalian interferon-induced, double-strandedRNA-activated protein kinase, (b) a reporter gene whose expression inincreased by activation of the protein kinase, and (c) a viral gene forproducing a viral inhibitor able to block activation of the proteinkinase. Preferably, the reporter gene is a fused GCN4/β-gal gene.

[0057] In a related aspect,. the yeast cell for use in screening agentseffective to inhibit viral replication in a host eukaryotic cell, wherethe virus activates or induces a cellular protein to interfere with theactivation of the host-cell interferon-induced, double-strandedRNA-activated protein kinase, includes the components (a) and (b) aboveand (c) a gene encoding a protein which blocks activation of a cellularprotein.

[0058] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF FIGURES

[0059]FIG. 1 shows the terminal stem, central domain, and apical stemloop of adenovirus VAI RNA (Ma, Y. and M. B. Mathews. 1993. Comparativeanalysis of the structure and function of adenovirus virus associatedRNAs. J. Virol. 67:6605-6617).

[0060]FIG. 2 shows the antisense VA (ava) oligodeoxynucleotide speciesava 1, ava 2, ava 3 and ava 9 annealed to complementary sequences of VAIRNA.

[0061]FIG. 3 shows the sequences of antisense species and complementaryVAI RNA regions, i.e., VAI RNA antisense oligodeoxynucleotides (ODN).

[0062]FIG. 4 shows the result of in vitro translation assay. Column 1:(−) mRNA; column 2: (+) mRNA; column 3: (+) mRNA, (+) reovirus dsRNA;column 4: (+) mRNA, (+) reovirus dsRNA, (+) VAI RNA. Columns 5-9: (+)mRNA, (+) reovirus dsRNA, (+) VAI RNA, and antisense as follows: column5: ava 1; column 6: ava 2; column 7: ava 3; column 8: ava 9; column 9:ava 15.

[0063]FIG. 5 shows human rhinovirus 14 5′ NTR sequence and predictedsecondary structure (Le, S.-Y., and Zuker, M. (1990) J. Mol. Biol. 216,729-741). The initiating AUG start codon for the polyprotein, atnucleotide (“nt”) 625, is shown as a shaded box, non-initiating AUGcodons are shown as clear boxes. The YnXmAUG motif found in allpicornavirus IRES elements and the 21-base conserved sequence found inall rhinovirus and enterovirus IRES elements are underlined. Nucleotidepositions on the rhinovirus genome are marked by numbers.

[0064]FIG. 6 shows a schematic diagram of mRNAs used for in vitrotranslation studies. A) bCRL mRNA containing the β-globin 5′ NTR drivingtranslation of the CAT reporter gene, and rhinovirus IRES drivingtranslation of the luciferase reporter gene. B) bL mRNA containing theβ-globin 5′ NTR driving translation of the luciferase reporter gene.Lines represent β-globin 5′ non-translated region (NTR), rhinovirusIRES, or 3′ NTRs, as indicated. Boxes represent reporter genes CAT(chloramphenicol acetyl transferase) and luciferase.

[0065]FIG. 7 shows in vitro translation of bLuc and bCRL mRNAs.Translation reactions were performed in duplicate as described by Lee,K. A. W., and Sonenberg, N. (1982) Proc. Natl. Acad. Sci. USA 79, 3447.Lane M, marker proteins; lanes 1-2, no mRNA; lanes 3-4, bL mRNA; lanes5-6, bL mRNA with anti-IRES-oligo; lanes 7-8, bCRL mRNA; lanes 9-10,bCRL mRNA with anti-IRES-oligo. Bands corresponding to luciferase andCAT translation products are indicated, along with protein markers of30, 46, and 69 kDa.

[0066]FIG. 8 shows luciferase activity assay of bL and bCRL mRNAtranslation reactions in the absence and presence of antisense(anti-IRES-oligo) and control (control-oligo) deoxyoligonucleotides.Translation reactions and luciferase activity assays were performed asdescribed in text. Relative light units from two independent replicateswere averaged and luciferase activity from bL and bCRL translationsnormalized to 100 for comparison. Translation reactions contained: lane1, no mRNA; lane 2, bL mRNA; lane 3, bL mRNA and anti-IRES-oligo; lane4, bL mRNA and control-oligo; lane 5, bCRL mRNA; lane 6, bCRL mRNA andanti-IRES-oligo; lane 7, bCRL mRNA and control-oligo.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Antiviral Agents

[0067] Given the large number of drugs available for treating infectionscaused by more complex organisms such as bacteria, it is remarkable howfew drugs are available for treating the relatively simple organismsknown as viruses. Indeed, most viral diseases remain essentiallyuntreatable. The major difficulty in developing anti-viral drugs isthat, unlike bacteria, viruses replicate inside host cells and utilizethe machinery of those cells for replication, sharing many nutritionalrequirements and synthetic pathways with their hosts. As a result, it isdifficult to identify agents that kill or arrest replication of a viruswithout also harming the host. Even those anti-viral drugs that havebeen approved for use in humans often have side effects which limittheir utility.

[0068] The majority of existing anti-viral drugs are nucleoside analogsor other agents that exert their effects through an enzyme involved inproducing new copies of the viral genetic material, such as a nucleosidekinase or a polymerase or reverse transcriptase or replicase. Theseanalogs are typically metabolized into nucleotide analogs that inhibitproduction of viral nucleic acid, for example by inhibiting a polymeraseor by causing premature chain termination of growing viral nucleicacids. The efficacy of such drugs depends on two key factors. The firstis that the target virus utilize: at least one virus-specific enzyme,encoded by the virus and used only by the virus, in the pathways whichresult in the copying of its genetic material. The second is that thisenzyme is more sensitive to the drug or more efficient in utilizing itthan any corresponding enzyme in the host. However, because viral andcellular nucleic acid metabolism are so similar, it is difficult to findanti-viral agents that are not used to some extent by host cell enzymes.This limits the dose of anti-viral drug that can be tolerated, which inturn may limit the utility of the drug.

[0069] Even in the case where a drug is tolerated at an effective dose,its effectiveness can be reduced markedly by the ability of a virus tomutate relatively rapidly, evolving new versions of the viral enzymewhich do not utilize the drug as efficiently or which are less inhibitedby the drug.

[0070] There is thus a clear need for novel anti-viral drugs that willbe effective at doses tolerated by the host and that will be moredifficult for viruses to evade by mutation.

[0071] The present invention provides novel methods for discovering suchdrugs and for treating illnesses with the drugs discovered. The methodsof this invention are based in the observation that many viruses takeover control of protein synthesis (translation of messenger RNA) incells they infect. The viral proteins are synthesized preferentiallyover host proteins in infected cells. This preferential synthesis ofviral proteins is important to the replication of the virus. Drugs whichreduce or prevent the viral takeover of protein synthesis are thereforeeffective anti-viral agents.

[0072] Such drugs have significant advantages over current anti-viralagents. As noted above, the targets for the majority of the latter areenzymes involved in the synthesis of viral nucleic acids, and becausehost cells also contain enzymes active in the synthesis of nucleic acidsit is difficult to hit the viral enzymes without also hitting the hostones. Similar problems are likely to occur for any drug target which isan active catalyst in the synthesis of a material required by both thevirus and the host cell. In the methods of the present invention, theseproblems are avoided because the drug targets are not active catalystsin a synthetic pathway: they are devices used by a virus to securepreferential access to a synthetic pathway (protein synthesis), ratherthan catalysts in such a pathway. As weapons used by the virus in itsattack on the host, these devices do not have any parallels within thehost. Drugs which interfere with these devices therefore have minimalside effects on the host.

[0073] Such drugs are more effective than current drugs, for tworeasons. First, their minimal side effects allow them to be used athigher doses. Second, it is possible for these drugs to be intrinsicallymore injurious to their targets than is tolerable for drugs whosetargets have host homologues, because if the latter drugs areintrinsically too injurious they may harm the host homologues to someextent.

[0074] Viruses are also less able to evolve resistance to drugs whichtarget viral translational hijacking devices. These devices must ofnecessity interact with host-cell components involved in proteinsynthesis, and the need to maintain these interactions means that thevirus is limited in the extent to which it can mutate its hijackingdevices. If it mutates too far to avoid a drug, it may no longer be ableto hijack protein synthesis. This limitation is particularly problematicfor the virus because it may need to make larger changes to evade anhijack-blocking drug than to evade a drug whose target is a syntheticenzyme with a host homologue, because, as noted above, thehijack-blocking drug may be intrinsically more injurious to its target.

[0075] In summary, the present invention provides a means to discoverand utilize novel anti-viral drugs with important advantages overcurrent such drugs, namely fewer side effects and a reduced likelihoodof the evolution of resistant viruses.

[0076] The methods of this invention are based in the observation thatmany viruses take control over the process of protein synthesis(translation of mRNA) in cells they infect. Viruses use a variety ofmechanisms to effect this takeover, including but not limited to the useof special viral nucleic acid sequences which ensure preferentialtranslation of viral RNAs (see e.g., Pelletier et al., Mol. Cell.Biol,8, 1103-1112, 1988; Trono et al. Science 241, 445-448; Sonenberg &Meerovitch, 1990; Garfinkel & Katze, J. Biol. Chem. 267, 9383-9390,1992), recruitment of cellular proteins to interact with these specialsequences (see e.g., Jang S K & Wimmer E, Genes Dev. 4, 1560-1572,1990), modification or degradation of host-cell components whichparticipate in translation or its control (see e.g., Katze MG et al., J.Virology 62, 3710-3717, 1988, Lee et al., Proc. Natl. Acad. Sci. USA 87,6208-6212, 1990), and disablement of cellular defenses mounted inresponse to the infection (see e.g., review by Katze M G, J. InterferonRes. 12, 241-248, 1992). Any such mechanism used by a virus to ensurepreferential translation of viral proteins as compared to host-cellproteins in infected cells can be addressed by the methods of thisinvention.

[0077] These methods are exemplified herein with descriptions of twosuch mechanisms used by viruses, namely (i) viral interference with ahost enzyme known by various names including p68 protein kinase and theinterferon-induced double-stranded RNA-activated protein kinase, and(ii) viral nucleic acid sequences responsible for preferentialtranslation of viral RNAs. The use of these examples is in no wayintended to limit the scope of the invention.

[0078] The protein known as p68 protein kinase is an interferon-induceddouble-stranded RNA-activated protein kinase. This kinase is activatedby the double-stranded RNA typically found in virus-infected cells. Onceactivated, the kinase phosphorylates the alpha subunit of the initiationfactor eIF-2, an event which quickly leads to a block in the initiationstage of translation. The effect is to shut down protein synthesis inthe cell, causing that cell to die: something which the multi-cellularinfected organism can afford but which the virus cannot. To ensurecontinued translation in infected cells, different viruses have evolveda variety of mechanisms to prevent or counteract activation of the p68kinase (reviewed in Katze, 1992). These include viral RNAs which bind tothe kinase and prevent binding of the double-stranded RNA activator, asused by adenovirus (reviewed by Mathews M B & Shenk T. J. Virology 65,5657-5662), HIV (Edery et al., Cell 56, 303-312, 1989; Gunnery et al.,Proc. Natl. Acad. Sci. USA 87, 8687-8691, 1990; Roy et al., J. Virology65, 632-640, 1991) and Epstein-Barr virus (Clarke et al., Eur. J.Biochem 193, 635-641, 1990; Clarke et al., Nucl. Acids Res. 19, 243-248,1991); viral proteins which bind the double-stranded RNA and prevent itbinding to the kinase, as used by vaccinia virus and reovirus (Watson etal., Virology 185, 206-216, 1991; Imani and Jacobs, Proc. Natl. Acad.Sci. USA 85, 7887-7891, 1988); viral proteins which act aspseudosubstrates of the kinase, as used by vaccinia virus (Beattie etal., Virology 183, 419-422, 1991); recruitment of a cellular protein,p58, to block activation of the kinase and inhibit active kinase, asused by influenza virus (Lee et al., 1990); and recruitment of acellular protein into a complex with RNA (possibly viral double-strandedRNA) which degrades p68, as used by poliovirus (Black et al., J.Virology, 67, 791-800, 1993).

[0079] p68, and the RNAs and proteins just described which interact withit, are examples of a broader class of macromolecules which have beenshown to be involved in the seizure or retention by viruses of controlof translation in infected cells. Other examples include: the hosttranslational factors eIF2 and eIF3/4B, which are reported to beimpaired in cells infected with vesicular stomatitis virus (VSV)(Centrella and Lucas-Lenard, J. Virology 41, 781-791, 1982; Thomas andWagner, Biochemistry 22, 1540-1546, 1983); the product of the VSV geneP, reportedly responsible for host translational inhibition (Stanners,et al., Cell 11, 273-281, 1977); the poliovirus 2A protease, responsiblefor degrading the p220 subunit of cap-binding protein complex (eIF-4F)in infected cells, and thereby preventing cap-dependent translation ofhost-cell mRNAs (Etchison et al., J. Biol. Chem. 257, 14806-14810,1982); the cellular protease recruited/activated by the poliovirusprotease 2A to cleave p220 (the poliovirus enzyme does not cleave p220directly) (Lloyd et al., Virology 150, 299-303, 1986); the p220 proteindegraded in poliovirus-infected cells; the host initiation factoreIF-4E, another component of the cap-binding protein complex which isdephosphorylated in adenovirus-infected cells to shut off host proteinsynthesis (Huang and Schneider, Cell 65, 271-280, 1991); and thecellular proteins p57 (also known as polypyrimidine tract-bindingprotein, pPTB), p50 and p52 implicated in the initiation of translationat internal ribosome entry sites within poliovirus and other viral RNAs(Jang and Wimmer, 1990; del Angel et. al., Proc. Natl. Acad. Sci. USA86, 8299-8303, 1989; Meerovitch et al., Genes Dev. 3, 1026-1034, 1989;Najita and Sarnow, Proc. Natl. Acad. Sci. USA 87, 5846-5850, 1990).

[0080] To these examples can be added a variety of macromolecules usedby viruses to cut off the supply of host-cell mRNAs and/or favor theproduction of viral RNAs in infected cells. These include: the vhs geneproduct of herpes simplex virus (HSV), a virion protein which degradesmRNAs in infected cells (Kwong and Frenkel, Proc. Natl. Acad. Sci. USA84, 1926-1930, 1987; Kwong et al., J. Virology 62, 912-921, 1988);another HSV virion protein which binds to a sequence-specificDNA-binding protein in host cells, causing increased transcription fromviral gene promoters (Campbell et al., J. Mol. Biol. 180, 1-19, 1984); acap-dependent endonuclease encoded by influenza virus which cleavesnascent host-cell transcripts in the nucleus to provide primers for thesynthesis of viral mRNA from the viral RNA genome (Bouloy et al., Proc.Natl. Acad. Sci. USA 75, 4886-4890, 1978; Plotch et al., Cell 23,847-858, 1981); nucleases used by influenza virus and poxvirus todegrade host-cell mRNAs (Rice and Roberts, J. Virology 47, 529-539,1983; Inglis S C, Mol. Cell. Biol 2, 1644-1648, 1982); viral inhibitorsof host-cell RNA polymerase II (the enzyme responsible for transcriptionof host-cell mRNAs) such as the “positive-strand leader RNA” believed tobind a host-cell factor and prevent its binding to host-cell promoters(Grinell and Wagner, J. Virology 48, 88-101, 1983); and adenovirusproteins E1B-55K and E4-34K which inhibit transport of host-celltranscripts from the nucleus to the cytoplasm (Babiss et al., Mol. Cell.Biol. 5, 2552-2558, 1985; Halbert et al., J. Virology 56, 250-257, 1985;Pilder et al., Mol. Cell. Biol. 6, 470-476, 1986).

[0081] The above are all examples of a broader class of macromoleculesinvolved in translation whose concentrations and/or activities aresubject to modulation by viruses. The present invention applies equallywell to other macromolecules within this broad class. A variety ofprocedures are available to those skilled in the art which enable themto identify other such macromolecules (including polypeptides, proteins,glycoproteins, lipids, carbohydrates, mucopolysaccharides, glycolipids,and nucleic acids), and to design methods for selecting compounds whichcan prevent or moderate the interaction between viruses and thesemacromolecules. In general, the steps required include: ascertainingwhether translation is modulated by a given virus during infection;identifying the specific macromolecule(s) mediating the effect ontranslation; identifying any other cellular and/or viral componentsinvolved; characterizing the interaction between these components; anddesigning a screening method in which disruption or moderation of thisinteraction can be detected. These steps can be performed in anysequence depending on the nature of the results obtained, and not allsteps may be required in order to select compounds which can have thedesired effect. The specific details of these steps now follow. Many ofthe procedures used are collected in such reference texts as Ausubel etal., (eds) Current Protocols in Molecular Biology, Wiley-Interscience,New York, 1991, and Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Ed.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

[0082] Determination That Virus Affects Translation

[0083] Several methods can be used to determine whether translation isaffected during infection by a particular virus. The overall rate ofprotein synthesis in infected and uninfected cells can be measured byincubating such cells in the presence of a labeled amino acid andmeasuring the incorporation of this labeled precursor into protein. Thelabeled amino acid may typically be one that includes a radioactiveisotope, such as [³⁵S]methionine, [³⁵ S]cysteine, [¹H]leucine, or[¹⁴C]leucine, and its utilization may typically be followed by measuringthe incorporation of radioactivity intotrichloroacetic-acid-precipitable protein. As well as the overall rateof protein synthesis, the rates of individual stages of translation,such as initiation and elongation, can be measured using standardprocedures such as polysome profiling and transit time determination. Asan alternative to incubating intact cells with radiolabeled substrates,extracts can be made from uninfected and infected cells and utilized inin vitro translations with these substrates, examining the translationof endogenous mRNAs or test mRNAs added to the cell extracts.

[0084] Additional important information about the effects of viruses ontranslation can be obtained by examining the types and relativequantities of proteins produced in uninfected and infected cells. Thiscan be achieved by incubating these cells in the presence ofradiolabeled amino acids as described above and then usingpolyacrylamide gel electrophoresis to separate the radiolabeled proteinsproduced. The separated proteins can be detected by autoradiography orwith a Phosphor Imager device, and analyzed by comparison with standardlabeled proteins of known molecular weights included on the samepolyacrylamide gel during electrophoresis. As in the case of ratedeterminations, these studies of protein synthesis can also be performedusing extracts made from cells rather than intact cells themselves.

[0085] Identification of Macromolecules

[0086] Once evidence is obtained that translation is affected byinfection with the virus under study, the activities and concentrationsof all macromolecules known to be involved directly or indirectly in theprocess of translation or its regulation can be compared in uninfectedand infected cells and/or in extracts made from such cells. If theevidence obtained points to an effect on a specific stage of translationsuch as initiation or elongation, attention might initially be directedbe directed to macromolecules known to be involved at that stage.

[0087] Macromolecules which may be examined include but are not limitedto known translation factors (reviewed and listed in Hershey, Ann. Rev.Biochem. 60, 717-755, 1991) such as initiation factors (such as eIF-1,eIF-1A, eIF-2, eIF-2A, eIF-2B, eIF-2C, eIF-3, eIF-3A, eIF-4A, eIF-4B,eIF-4F (p220), eIF-5 and eIF-5A) elongation factors (such as eEF-1a,eEF-1b, eEF-1g and eEF-2), termination factors (such as eRF), ribosomalproteins, kinases and phosphorylases and any other enzymes which actdirectly or indirectly to modify any of the proteins just listed or anyother macromolecules involved in translation, proteases which maydegrade any proteins important for translation, ribosomal RNAs (rRNAs),transfer RNAs (tRNAs), enzymes which synthesize or degrade rRNAs andtRNAs, aminoacyl-tRNA synthetases, interferons, and any othermacromolecules which induce or repress the synthesis of components orregulators of the translational apparatus.

[0088] Methods which can be used to analyze components involved intranslation include, but are not limited to, functional assays of enzymeactivity, in vitro translations, coupled in vitrotranscription-translation reactions, incubations with [gamma-³²P]ATP toallow determination of phosphorylation status, immunoprecipitation,one-dimensional and two-dimensional gel electrophoresis, Westernblotting, differential centrifugation, chromatographic purification,UV-crosslinking, gel retardation assays, other DNA-binding andRNA-binding assays, and the like.

[0089] Another approach to identifying a component involved in a viraleffect on translation is to make extracts from uninfected and infectedcells and fractionate these extracts based on their ability to exhibitan effect on in vitro translation reactions. Thus, extracts fromuninfected and infected cells are initially added to parallel butseparate in vitro translation reactions, and their effects on thesereactions compared. The two types of extract are then be fractionated inparallel using a variety of procedures known to those skilled in theart, and corresponding fractions from the two extracts are tested inparallel for their effects on in vitro translation reactions. Fractionsfound to contain a translation-affecting component from infected cellsare then fractionated further in parallel with the correspondingfractions from uninfected cells, and the new fractions obtained fromthis next round of fractionation are also tested in in vitro translationreactions. Repeated iterations of this fractionation and testingprocedure eventually provides a relatively purified fraction frominfected cells which contains the component (s) involved in the observedviral effect on translation.

[0090] Fractionation methods which can be used in this approach include,but are not limited to, centrifugation, ammonium sulfate precipitation,other differential precipitations, gel filtration, ion exchangechromatography, hydrophobic interaction chromatography, reverse phasechromatography, affinity chromatography, differential extractions,isoelectric focusing, electrophoresis, isotachophoresis, and the like.

[0091] Since translation depends on the availability of mRNA templates,it may also be important to extend the analyses to cover the synthesis,processing, transport and degradation of mRNA. mRNA synthesis(transcription) can be examined in an manner analogous to proteinsynthesis, by utilizing the incorporation of labeled precursors intomRNA in order to determine overall rates of mRNA synthesis and togenerate labeled material that can be examined by gel electrophoresis,in this case on agarose as well as polyacrylamide gels. Processing andtransport of mRNA can also be examined using labeled precursors, toanalyze the sizes and quantities of various labeled RNA species innuclear and cytoplasmic extracts of cells. Alternatively, the sizes andquantities of these RNAs can be examined by the Northern blothybridization procedure, in which RNAs that have been separated byelectrophoresis and transferred to a hybridization membrane are detectedby hybridization with a labeled nucleic acid probe specific for the RNAsof interest. Degradation of mRNAs can be followed by similar procedures,using radiolabeled mRNAs or Northern blot hybridizations to trace thefate of mRNAs. For all stages of mRNA synthesis, processing, anddegradation it may also be useful to measure the activities andconcentrations of the enzymes and other proteins involved, such as RNApolymerases, splicing enzymes, splice-junction binding proteins, andribonucleases responsible for degrading mRNAs. Alterations intranscriptional activity may also be detected and analyzed utilizingcell extracts for in vitro transcription reactions.

[0092] Identification of Other Components Involved

[0093] Detailed investigation of viral effects on translation may oftenreveal one or more cellular components whose activity or concentrationis modulated during viral infection. It might, for example, identify aninitiation factor or elongation factor or subunit thereof which isdegraded in infected cells, or which becomes phosphorylated,dephosphorylated or otherwise modified in a way that alters itsactivity. Once one such effect has been observed, it points the way forfurther investigations to identify additional cellular and/or viralcomponents involved.

[0094] Thus, if a component of the translational apparatus has beenfound to be degraded, attention may turn to identifying the enzymeresponsible for this degradation. This is achieved by measuring theactivities of enzymes known to act upon the degraded component, or byfractionating extracts from infected and uninfected cells and measuringthe component-degrading activity of each fraction. Repeated rounds offractionation by a variety of procedures known to those skilled in theart can be used to isolate the degrading activity. Fractionationprocedures which may be used include, but are not limited to,centrifugation, ammonium sulfate precipitation, other differentialprecipitations, gel filtration, ion exchange chromatography, hydrophobicinteraction chromatography, reverse phase chromatography, affinitychromatography, differential extractions, isoelectric focusing,electrophoresis, isotachophoresis, and the like.

[0095] A similar approach can be adopted if the observation is made thata component of the translational apparatus undergoes phosphorylation,dephosphorylation or other modification during viral infection. Thus,measurements may be made of the activities of enzymes known to performsuch modifications on the component in question, or extracts fromuninfected and infected cells may be fractionated to isolate theenzyme(s) responsible, testing each fraction for its ability to modifythe component in the manner originally observed.

[0096] Inhibitors of a given translational step or component maylikewise be identified by fractionating extracts from uninfected andinfected cells and testing each fraction for its ability to inhibit thestep or component in question.

[0097] Translation-affecting components isolated by any of theaforementioned fractionation approaches can be utilized to help clonethe gene(s) which code for these components. If, for example, thecomponent isolated is a protein, its amino acid sequence or a part ofthat sequence can be determined by well known protein sequencingmethods, and the sequence information obtained can be used to predictthe sequence of oligonucleotides which can be used as reversetranscriptase primers for cDNA synthesis or as amplification primers forthe polymerase chain reaction, or as hybridization probes for screeninggene/cDNA libraries. Alternatively, the isolated component can be usedas an immunogen to raise antibodies against the component, whichantibodies can then be used to screen cDNA expression libraries toidentify clones encoding the component. Antibodies can also be raised bysynthesizing a short peptide corresponding to part or all of any aminoacid sequence determined from the isolated component, and using thispeptide as immunogen. The peptide-induced antibodies can be used toscreen cDNA expression libraries, or to affinity-purify the component inlarger quantities enabling more extensive sequence determination, andthus providing more extensive information on which to base a cloningstrategy.

[0098] The identification of viral components responsible for effects ontranslation may be facilitated by examining mutant viruses, eithernaturally occurring mutants or mutants made in the laboratory. Thelatter may be constructed by a variety of procedures known to thoseskilled in the art, including but not limited to, chemical treatmentwith mutagens, and the use of molecular biology techniques to generateinsertions, substitutions, deletions and point mutations in viral genesor the viral genome. The impact of various mutations on the interactionsbetween the virus and host-cell translation can then be assessed. Ifparticular mutations alter or abolish the effect(s) which a virus has ontranslation, this provides strong evidence that the gene or genes inwhich the mutations occur are important in mediating these effects.

[0099] Further evidence for the involvement of these genes and theirproducts can be obtained in a variety of ways. One route is to userecombinant DNA techniques to produce the product(s) of the viralgene(s) implicated by the mutational analysis, and then to test theeffects of these gene products on translation. The testing can beperformed, for example, by adding the viral gene products to in vitrotranslation reactions or by expressing these gene products in intactcells.

[0100] Even without mutational analysis, in vitro transcription andtranslation procedures can be used to determine whether the addition ofviral genomes or RNAs or subsets or fragments thereof, or thetranslation products of such molecules, has an impact on translation.Such genomes or RNAs or subsets or fragments can be obtained in avariety of ways, for example, by purification from virus particles,extraction from infected cells, cleavage of intact viral RNAs or DNAsusing ribonucleases or deoxyribonucleases, oligodeoxynucleotide-directedcleavage of viral RNAs by ribonuclease H, cleavage of viral DNAs byrestriction endonucleases, amplification of specific segments of viralRNA or DNA by the polymerase chain reaction, transcription from clonedviral genes or cDNAs, and so on.

[0101] Viral components involved in effects on translation can also beidentified by introducing individual viral components or genes/cDNAswhich encode them or fragments of components or genes/cDNAs into intactcells rather than in vitro translation reactions. The translationalstatus in cells into which such an introduction had been made can thenbe compared with the status within cells which had received a “mockintroduction” or none at all. A change in translational status wouldimplicate the viral component or gene/cDNA or fragment thereof which hadbeen introduced into the cell.

[0102] Another approach to identifying cellular and viral componentsinvolved in translational effects is to use labeled nucleic acidsprepared from uninfected and infected cells as probes in differentialhybridization screens of gene/cDNA “libraries” made from viral orcellular nucleic acids. Such libraries are often made in the Lambda gt10vector or similar vectors. Clones which behave differently towards thelabeled nucleic acid probes from infected and uninfected cells will beinvestigated further, since they represent sequences whose hybridizationpartners are either more abundant or less abundant in infected cellsthan in uninfected cells.

[0103] In a modification of this approach, labeled proteins rather thannucleic acids can be prepared from uninfected and infected cells, andthe differential screening can be performed under conditions which favorprotein-nucleic acid interactions. In this case, clones which behavedifferently towards the labeled protein probes represent sequences whichare partners for nucleic acid-binding proteins that are either moreabundant or less abundant in infected cells than in uninfected cells.

[0104] A similar approach can be adopted utilizing expression librariesmade from viral or cellular nucleic acids, that is, libraries made insuch a way that the protein encoded by each cloned gene is expressedwithin the clone that contains it. Such libraries are often made in theLambda gt11 vector or similar vectors. In this case, differentialscreening with labeled nucleic acids from uninfected and infected cellswill reveal clones encoding proteins that interact with nucleic acidswhich are either more abundant or less abundant in infected cells thanin uninfected cells. Differential screening with labeled proteins on theother hand will reveal clones encoding proteins that interact with otherproteins which are either more abundant or less abundant in infectedcells than in uninfected cells.

[0105] Proteins involved in important interactions with other proteinscan also be identified using a yeast genetic system known as thetwo-hybrid system (Fields & Song, Nature, 340, 245-246, 1989; Chien etal., Proc. Natl. Acad. Sci. USA 88, 9578-9582, 1991). This requires theavailability of a gene or cDNA encoding one of the two proteins whichinteract with each other. In the present case this gene or cDNA can beobtained by any of the several methods described in the preceding text.This gene or cDNA is cloned into a specific plasmid in such a way thatit is expressed fused to the DNA-binding domain of a yeasttranscriptional activator such as GAL4 which has two separable andfunctionally essential domains, one for DNA-binding and the other fortranscriptional activation. In parallel, genes or cDNAs encodingputative binding partners of the known component are cloned in such away that each putative partner is expressed fused to the transcriptionalactivation domain of the same DNA-binding protein. Introduction of bothtypes of fusion into the same yeast cell results in generation offunctional DNA-binding protein only if the fusion partners of the twodomains of this protein interact with one another closely enough tobring together its two separately-expressed domains. clones whichproduce such functional DNA-binding protein can be selected very easilyby plating them on a medium which requires the yeast to produce anenzyme that is under the control of the. DNA-binding protein. The geneor cDNA for the partner which binds to the previously identifiedcomponent can then be recovered from yeast clones which grow on theselective medium.

[0106] Many other methods are available for further investigation of aninitial observation that some component involved in translation ismodulated in infected cells. Other options include but are not limitedto: using the component in question as an affinity ligand to identifyviral and cellular products which bind to it; labeling this componentwith a detectable label and using it as a probe to detect viral andcellular products on blots of electrophoresis gels; labeling thecomponent and using it to probe libraries of viral and cellular genesand/or cDNAs; labeling the component and using it to probe cDNAexpression libraries to find clones synthesizing proteins which can bindto the component; performing UV-crosslinking studies to identify viralor cellular products which can bind to the component; using thecomponent in gel retardation assays which would detect its ability tobind to viral or cellular nucleic acids; performing footprintinganalyses to identify the regions within a nucleic acid to which thecomponent binds; and so on.

[0107] From this description it should be evident that a wide variety ofmethods is available to someone skilled in the art to identify viral andcellular components which interact with a component that has been foundto be modulated in viral-infected cells.

[0108] Interactions Between Components

[0109] Many different methods are available to characterize theinteractions between cellular and viral components which affecttranslation. The susceptibility of such an interaction to changes in pH,ionic strength, temperature, the nature and mixture of anions andcations present, the relative concentrations of the two components, theabsolute concentrations of these components, the availability ofcofactors, the availability of an energy source, the presence or absenceof lipids, of nucleic acids, of carbohydrates, of other proteins, and/orof any other additives can all provide information about the nature ofthe interaction between the components. So too can the susceptibility ofthe interaction to treatment of one or both components with alkylatingagents, oxidizing agents, reducing agents, or other agents which causechemical modifications, or with enzymes that phosphorylate,dephosphorylate, glycosylate, deglycosylate, add lipid side-chains,remove lipid side-chains, or cause other enzymatic modifications, bemeasured.

[0110] Also informative are the effects of truncations, additions,substitutions, deletions, inversions and point mutations in one or bothcomponents. Such structurally altered components can be generated bytreatment of intact components with cleavage enzymes such as proteases,endoribonucleases and endodeoxyribonucleases, with editing enzymes suchas DNA polymerases, with joining enzymes such as RNA ligases, DNAligases, and RNA splicing enzymes, with copying enzymes such as DNApolymerases, RNA polymerases, and reverse transcriptases, withend-specific degrading enzymes such as 5′-exonucleases, 3′-exonucleases,aminopeptidases and carboxypeptidases, with enzymes that can addextensions to ends such as terminal deoxynucleotidyl transferase andpoly(A) polymerase, and so on. Alternatively, structurally alteredcomponents can be generated by making appropriate alterations to clonedgenes and expressing these genes in intact cells or in in vitro systems.Thus, the use of restriction enzymes, ligases, linkers, adaptors,reverse transcriptases, DNA polymerases, RNA polymerases, polymerasechain reactions, site-directed mutagenesis, and randomized mutagenesismake it possible to generate an enormous spectrum of structurallyaltered forms of components which interact with one another. Thesestructural alterations can then be tested in the array of methodspreviously described to determine whether the alterations change orabolish the interaction between different components and/or the impactof these components on translation.

[0111] Methods to Screen Potential Agents

[0112] Methods to screen potential agents for their ability to disruptor moderate viral effects on translation can be designed withoutdetailed knowledge of the precise interaction between viral and cellularcomponents, although such a knowledge can certainly be helpful. Inprinciple, many of the numerous methods which have so far been describedto identify viral and cellular components involved in effects ontranslation can be readily adapted to detect interference with theinteraction between these components. Thus, for example, if it has beenfound that viral infection leads to the phosphorylation,dephosphorylation or other modification of a given component, or to achange in its catalytic activity such as the inhibition of thatactivity, or to enhanced synthesis or degradation of this component, orto any other observable effect described in the foregoing disclosure,then agents can be screened for their ability to prevent or moderatethis effect on the component in question. The screening can be performedby adding the test agent to intact cells which have been infected byvirus and then examining the component of interest by whatever procedurehas been established to demonstrate the viral effect on this component.Alternatively, the screening can be performed by adding the test agentto in vitro translation reactions and then proceeding with theestablished analysis. As another alternative; purified or partiallypurified components which have been determined to interact with oneanother by the methods described above can be placed under conditions inwhich the interaction between them would normally occur, with andwithout the addition of the test agent, and the procedures previouslyestablished to analyze the interaction can be used to assess the impactof the test agent. In this approach, the purified or partially purifiedcomponents may be prepared by fractionation of extracts from uninfectedand infected cells, or they may be obtained by expression of clonedgenes or cDNAs or fragments thereof, optionally followed by purificationof the expressed material.

[0113] Within the broad category of in vitro selection methods, severaltypes of method are likely to be particularly convenient and/or usefulfor screening test agents. These include but are not limited to methodswhich measure a binding interaction between two or more components,methods which measure the activity of an enzyme which is one of theinteracting components, and methods which measure the activity orexpression of “reporter” protein, that is, an enzyme or other detectableor selectable protein, which has been placed under the control of one ofthe components.

[0114] Binding interactions between two or more components can bemeasured in a variety of ways. One approach is to label one of thecomponents with an easily detectable label, place it together with theother component(s) in conditions under which they would normallyinteract, perform a separation step which separates bound labeledcomponent from unbound labeled component, and then measure the amount ofbound component. The effect of a test agent included in the bindingreaction can be determined by comparing the amount of labeled componentwhich binds in the presence of this agent to the amount which binds inits absence.

[0115] The separation step in this type of procedure can be accomplishedin various ways. In one approach, (one of) the binding partner(s) forthe labeled component can be immobilized on a solid phase prior to thebinding reaction, and unbound labeled component can be removed after thebinding reaction by washing the solid phase. Attachment of the bindingpartner to the solid phase can be accomplished in various ways known tothose skilled in the art, including but not limited to chemicalcross-linking, non-specific adhesion to a plastic surface, interactionwith an antibody attached to the solid phase, interaction between aligand attached to the binding partner (such as biotin) and aligand-binding protein (such as avidin or streptavidin) attached to thesolid phase, and so on.

[0116] Alternatively, the separation step can be accomplished after thelabeled component had been allowed to interact with its bindingpartner(s) in solution. If the size differences between the labeledcomponent and its binding partner(s) permit such a separation, theseparation can be achieved by passing the products of the bindingreaction through an ultrafilter whose pores allow passage of unboundlabeled component but not of its binding partner(s) or of labeledcomponent bound to its partner(s). Separation can also be achieved usingany reagent capable of capturing a binding partner of the labeledcomponent from solution, such as an antibody against the bindingpartner, a ligand-binding protein which can interact with a ligandpreviously attached to the binding partner, and so on.

[0117] Test methods which rely on measurements of enzyme activity areperformed in accordance with the characteristics of the enzyme in eachcase. As noted above, a variety of enzyme activities can be determinedto be involved in the translational effect of a virus, including but notlimited to kinases, phosphatases, glycosylases, deglycosylases,transferases, lipases, deoxyribonucleases, ribonucleases, proteases,synthetases, polymerases, and the like, as well as those other enzymeactivities noted above. In general, measurements of enzyme activityrequire the ability to measure the product of the reaction in thepresence of other materials, and often to distinguish or separate theproduct of the reaction from the substrate for the reaction. Methodswhich may be used to measure reaction products include but are notlimited to measurement of the transfer or incorporation of a radioactiveor other labeled atom or group, spectrophotometric or colorimetricmeasurement of the concentration of the product, measurement of lightoutput from a luminescent or chemiluminescent reaction, measurement offluorescence from a fluorescent product, immuno-assays, otherimmunochemical procedures, and other competitive binding assays. Enzymeactivity can also be measured using any of the procedures just mentionedto detect the product of a secondary reaction or reactions which rely onthe product of the reaction of interest as a substrate or a cofactor.

[0118] In many cases the product of an enzyme reaction can be detectedwithout separating that product from other constituents of the reactionmixture, as for example when an uncolored chromogenic substrate givesrise to a colored product or the absorption spectrum for the product isdifferent from that of the substrate, allowing selection of a wavelengthfor absorbance measurements of just the product. Immunoassays, otherimmunochemical procedures and other competitive binding assays can alsooften be performed without first separating the product of interest.

[0119] In other cases it may be necessary to include a separation stepor steps to separate the product from other constituents of the reactionmixture before measuring it. In such cases, the necessary separation canbe accomplished by a variety of procedures, including but not limited tocentrifugation, trichloroacetic acid precipitation, ethanolprecipitation, ammonium sulfate precipitation, other differentialprecipitations, gel filtration, ion exchange chromatography, hydrophobicinteraction chromatography, reverse phase chromatography, affinitychromatography, differential extractions, isoelectric focusing,electrophoresis, isotachophoresis, and the like.

[0120] In addition to methods which measure the activity of an enzymeimplicated in a viral effect on translation, test methods may also beemployed which have been configured such that the component(s)implicated in the viral effect controls the activity or expression of a“reporter” protein, that is, an enzyme or other detectable or selectableprotein. In the case, for example, where a kinase has been implicated inthe viral effect, the test method might be configured in such a way thatphosphorylation of a particular protein by the kinase leads to theactivation or inhibition of that protein or of some other proteincontrolled by that protein. In yeast, for example, phosphorylation ofeIF2-α by the GCN2 protein (or by mammalian p68 kinase substituting forGCN2) leads to an inhibition of the initiation of translation, which inturn leads to an increase in the synthesis of the GCN4 protein, which inturn induces the synthesis of further proteins involved in amino acidbiosynthesis. “Reporter” proteins can be readily fused to the GCN4protein at the genetic level so that the synthesis of these reporters iseffectively induced by the initial phosphorylation event catalyzed byGCN2 or mammalian p68.

[0121] Similar approaches can be used to detect modulation by testagents of the activity of a variety of other components which might beimplicated in viral effects on translation. The effect of a test agenton a protease, for example, can be monitored by following the survivalin an in vitro reaction of a reporter protein which is a target for thatprotease. Similarly, the effect of a test agent on a nuclease can bemonitored by following the appearance in an in vitro translationreaction or in vitro transcription-translation reaction of a reporterprotein translated from a suitably configured coding sequence providedto the reaction.

[0122] Proteins suitable for use as reporters in such assays include,but are not limited to, easily assayed enzymes such as β-galactosidase,luciferase, β-glucuronidase, chloramphenicol acetyl transferase, andsecreted embryonic alkaline phosphatase; proteins for which immunoassaysare readily available such as hormones and cytokines; proteins whichconfer a selective growth advantage on cells such as adenosinedeaminase, amino-glycoside phosphotransferase (the product of the neogene), dihydrofolate reductase, hygromycin-B-phosphotransferase,thymidine kinase (when used with HAT medium), xanthine-guaninephosphoribosyltransferase (XGPRT), and proteins which provide abiosynthetic capability missing from an auxotroph; proteins which confera growth disadvantage on cells, for example enzymes that convertnon-toxic substrates to toxic products such as thymidine kinase (whenused with medium containing bromodeoxyuridine) andorotidine-5′-phosphate decarboxylase (when used with 5-fluorooroticacid); and proteins which are toxic such as ricin, cholera toxin ordiphtheria toxin.

[0123] Many of the methods so far described for selecting test agentshave involved examining the impact of these agents on the interactionbetween two or more components in in vitro reactions. The interactingcomponents can also be brought into contact with one another withincells rather than in in vitro reactions. In this approach, codingsequence(s) encoding part or all of a component or components would beintroduced into a selected type of cell. Coding sequences for thisapproach include cloned genes or cDNAs or fragments of either orfragments amplified by the polymerase chain reaction or natural RNAs ortranscribed RNAs or the like. Several variations of the approach arepossible. In one variation, a coding sequence is introduced for a firstcomponent into a cell known to contain components with which this firstcomponent will interact. Thus, for example, a coding sequence for aviral component is introduced into a cell which is a normal target forinfection by the virus in question. Agents are tested to select thosewhich block the effect of the viral component within the cell into whichthe coding sequence has been introduced. In another variation, codingsequences for two or more components which interact with one anothermight be introduced into a cell, and agents tested for their ability tomoderate the interaction between these components, this interactionbeing followed by the procedures previously established as suitable forthe purpose. The cell into which the coding sequences are introduced canbe one which would normally be a target for infection by the virus inquestion. Alternatively and usefully, the cell can be one which iseasier to grow, manipulate and test such as a yeast cell. Indeed, thereare distinct advantages to reconstructing a translation controlmechanism in heterologous cells, in which the interactions between thecomponents involved are easier to study than they are when thosecomponents are in their normal environment. In the case of yeast, inparticular, the powerful genetic approaches available often make itpossible to identify and isolate the yeast homologues of genes fromhigher eukaryotes more quickly than the corresponding genes can beidentified in the higher eukaryotes.

[0124] From the foregoing it should be apparent that one skilled in theart is able to choose from a wide variety of methods at each stage inthe identification of components involved in viral effects ontranslation, in the characterization of the interaction between thesecomponents, and in the implementation of screening tests to selectcompounds which moderate or abolish the interaction between thesecomponents.

[0125] Protein Kinase

[0126] The following is a more detailed outline of the specificscreening and related protocols useful in this invention. This sectiondescribes a method for screening agents effective to inhibit viralreplication in a host eukaryotic cell. As one detailed example, thesystem chosen is one in which the virus is able to produce a viralinhibitor which interferes with the activity of the host-cellinterferon-induced, double-stranded RNA-activated protein kinase. Asnoted above, however, this example is not limiting in the invention andonly exemplifies the broad scope of the invention.

[0127] The method generally includes the steps of incubating the proteinkinase, the viral inhibitor, and the compound to be tested, underconditions effective to cause viral inhibitor interference with theactivation of the protein kinase, and examining the mixture forinterference. The invention contemplates four general embodiments, asdetailed below.

[0128] The particular screening protocol will depend to some extent onthe site of action of the virus inhibitor. For example, various virusesdegrade the kinase (e.g., polio), others inhibit activation of thekinase (Adenovirus VA1 RNA, Epstein-Barr virus EBER-1, HIV-1 TAR RNA,and Influenza), yet others bind dsRNA (Reovirus sigma 3 and vacciniaSKIF (E3L)), and others inhibit activity of the kinase (Influenza, SV40Tag, and Vaccinia K3L). These various mechanisms can be attacked bydifferent inhibitory agents of this invention which can be identified bymethods described below.

[0129] A. In vitro Screening for Compounds

[0130] In one example, the method is used for screening a compoundeffective to inhibit replication in a host cell of a virus whichproduces a viral inhibitor able to bind to the p68 protein kinase andblock its activation by double-stranded RNA (dsRNA). Here, theincubating step includes incubating the mixture under conditionseffective to bind the viral inhibitor to the protein kinase, and theexamining step includes examining the protein kinase for bound viralinhibitor.

[0131] The incubating may be carried out, for example, in solutionphase, and the examining step includes passing the mixture through afilter which retains the viral inhibitor only when the inhibitor isbound to the protein kinase.

[0132] Alternatively, the protein kinase may be bound to a solidsupport, the viral inhibitor labeled with a reporter, and the examiningstep performed by measuring the amount of reporter bound to the solidsupport.

[0133] Alternatively, the incubating may be carried out under conditionsin which the protein kinase is autophosphorylated, in the absence ofbinding to the viral inhibitor, and the examining step performed bydetermining the extent of phosphorylation of the p68 kinase.

[0134] In a second example, the incubating step includes incubating themixture under conditions effective for the p68 kinase to be activated inthe absence of the viral inhibitor, and the examining step includesexamining the activity of the p68 kinase in the presence of theinhibitor.

[0135] The incubating may be carried out, for example, using a purifiedor partially purified p68 kinase preparation, and the examining stepincludes measuring autophosphorylation of the kinase or phosphorylationof eIF2-alpha or histone substrates provided to the kinase.

[0136] Alternatively, the incubating may be carried out in an in vitrotranslation mixture containing the p68 kinase, and the examining stepincludes measuring the amount of a reporter polypeptide produced bytranslation of specific mRNA. The mRNA may be one whose translation isreduced by activation of p68 kinase, or preferably, one whosetranslation is increased, such as a chimeric RNA whose 5′-untranslatedleader is derived from the yeast GCN4 gene.

[0137] In a third example, the method is used for screening compoundseffective in blocking viral replication of a virus which produces aviral inhibitor effective to activate a host-cell component which isable in activated form to block activation of the protein kinase orinhibit the activated kinase. Here the mixture formed includes thehost-cell component (in activated form), the incubating step is carriedout under conditions in which the protein kinase is activated, in theabsence of the activated component, and the examining step includesexamining the mixture for inhibition of protein kinase activity.Alternatively, the mixture formed includes the host-cell component innon-activated form and the viral inhibitor which activates the host-cellcomponent, the incubating step is carried out under conditions in whichthe protein kinase is activated, in the absence of the viral inhibitorand activated host-cell component, and the examining step includesexamining the mixture for inhibition of protein kinase activity

[0138] B. In vivo Screening

[0139] In a fourth example, the viral or virus-activated inhibitor isexpressed in a yeast cell which is constructed to increase theexpression of a reporter polypeptide in the presence of activated p68kinase, and the examining step includes examining the yeast cells forincreased expression of the reporter polypeptide.

[0140] One of the yeast proteins which participates in translationcontrol is the protein GCN2. The protein is a kinase which is activatedby binding of uncharged tRNAs, which accumulate when amino acids are inshort supply. The activated protein inhibits translation levels inyeast, by phosphorylating the alpha subunit of the initiation factoreIF2. Another result of GCN2 activation is increased production of ayeast GCN4 protein, which then activates anabolic pathways for thesynthesis of amino acids.

[0141] A construct used in the present invention for in vivo screeningis a yeast cell in which the GCN2 gene is replaced with a mammalian p68gene under the control of a regulated promoter. The cell also includesthe additional modifications described below. Introduction of the p68gene into yeast can be carried out using standard recombinant techniquesfor introducing a selected coding sequence into yeast. Briefly, the p68gene is placed under the control of a down-regulatable promoter, withcell selection occurring under down-regulated conditions. This is donebecause in yeast cells the p68 protein is constitutively activated,presumably by endogenous dsRNA, and if expressed at too high a level itinhibits cell translation in its activated condition.

[0142] The yeast cells are then further constructed to enable theregulation of p68 to be tested by examining the levels of a reporterpolypeptide whose production is dependent on the presence of activatedp68 enzyme. Such a reporter can be produced from a β-gal gene fused tothe GCN4 yeast gene. The latter gene becomes expressed under conditionsof GCN2 activation, and has been shown to be under the control of thep68 phosphorylation system in yeast cells in which GCN2 has beenreplaced with p68. Thus, the presence of activated p68 leads to ashutdown of yeast translation in general, but to enhanced production ofthe fused GCN4/β-gal protein. The expression of the fused protein can bemeasured easily by measuring β-gal activity.

[0143] The screening system is designed for screening drugs which areeffective to disrupt a viral pathogen's counter-defense against the hostcell's attempt to shut down cell translation, by activation of the p68protein. The viral counter-defense may include, among others, (a) a VA1,EBER-1, or TAR viral inhibitor RNA which occupies the binding site onp68 and prevents dsRNA from binding to and activating p68, or (b) theability of the virus, e.g., influenza virus, to induce or activate acellular component which is effective to prevent activation of p68 ordeactivate the activated enzyme, or (c) a viral protein such as reovirusσ3 protein or vaccinia virus K3L and E3L proteins which blocks theactivation or activity of p68, or (d) a complex of a cellular componentwith a viral RNA, such as the complex used by poliovirus to degrade thep68 kinase.

[0144] In the case of a viral inhibitor, the yeast cells used inscreening are further constructed to contain the gene for the viralinhibitor under the control of an inducible promoter. Under non-inducinggrowth conditions, sin which the viral inhibitor is not expressed (butp68 protein is), the p68 protein is activated, presumably by endogenousdsRNA as noted above, and the presence of activated p68 is manifested byrelatively high measured levels of the GCN4/β-gal fusion protein. Underinducing growth conditions, for example, when the growth medium includesthe inducer for the inducible promoter which controls the expression ofthe viral inhibitor, the cells show low levels of activated p68 due tothe presence of the viral inhibitor, and this is manifested byrelatively low levels of the GCN4/β-gal fusion protein. Potentialantiviral agents are tested by assessing their impact on the measuredlevels of the GCN4/β-gal fusion protein under inducing conditions forthe viral inhibitor. Those agents which allow relatively high levels offusion protein to be synthesized are selected, as being agents whichprevent the viral inhibitor from interfering in activation of p68 byendogenous double-stranded RNA.

[0145] In the case of a cellular component induced or activated by avirus to prevent activation of p68 kinase or inhibit activated kinase,the gene for this cellular component is placed in the yeast cells usedfor screening under the control of an inducible promoter (in place ofthe viral inhibitor RNA gene described above). The yeast strain is thenused for screening essentially as described for viral inhibitors. Thus,under non-inducing growth conditions, the cellular component is notexpressed, and relatively high levels of the GCN4/β-gal fusion proteinare observed, reflecting the presence of p68 activated by endogenousdouble-stranded RNA. Under inducing growth conditions, the levels of theGCN4/β-gal fusion protein are lower, reflecting inhibition of theactivation or activity of p68 by the cellular component. Potentialantiviral agents are tested by assessing their impact on the measuredlevels of the GCN4/β-gal fusion protein under inducing conditions forthe cellular component, and agents selected which allow relatively highlevels of fusion protein to be synthesized.

[0146] A similar approach is adopted in the case of a complex between acellular component and a viral component which degrade the p68 kinase.In this case, the yeast strain would be further constructed to containgenes for both the cellular and the viral component under induciblecontrol, and the screening would be performed essentially as describedabove.

[0147] The following examples illustrate the screening methods describedabove, but in no way are intended to limit the scope of the invention.

EXAMPLE 1 Preparing p68 Protein Kinase

[0148] A. From Interferon-Induced Human Cells

[0149] p68 protein kinase is prepared from interferon-induced humantissue culture cell lines. Cells are lysed by Dounce homogenization, andnuclei and cell debris removed by centrifugation at 30,000×g for 20minutes. 4 M KCl is added to the supernatant to a final concentration pf100 mM, and ribosomes are pelleted by centrifugation at 60,000 rpm inBeckman type 60 rotor. The ribosomal pellet is resuspended in 800 mMKCl, 20 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT and 1 μMphenylmethylsulfonyl fluoride (PMSF), then homogenized using a Douncehomogenizer. The ribosomes are then centrifuged again at 60,000 rpm for90 min at 4° C. in a type 60 rotor. The resulting supernatant isdialyzed against 50 mM KCl, 20 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 0.1 mMEDTA, 1 mM DTT, 10% glycerol and 1 μM PMSF. The dialysate is centrifugedagain to remove solids. The resulting supernatant. (ribosomal salt wash)is applied to a DEAE-cellulose column equilibrated in 50 mM KCl, 20 mMHEPES (pH 7.4), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 10% glycerol and 1μM PMSF. p68 kinase is collected in the flow through fraction, adjustedto pH 6.8, and applied to a S-Sepharose Fast Flow (Pharmacia) columnequilibrated with 50 mM KCl, 20 mM HEPES (pH 6.8), 1.5 mM MgCl₂, 0.1 mMEDTA, 1 mM DTT, 10% glycerol and 1 μM PMSF. p68 kinase is eluted fromthe column in a linear gradient of 50-500 mM KCl in 20 mM HEPES (pH7.4), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 10% glycerol and 1 μM PMSF.The p68 kinase peak is loaded onto a hydroxyapatite HPHT (BioRad) columnequilibrated in 50 mM KCl, 20 mM HEPES (pH 7.2), 50 mM potassiumphosphate (pH 7.2), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 10% glyceroland 1 μM PMSF. p68 kinase is eluted in a linear gradient of 50-500 mMpotassium phosphate (pH 7.2). The p68 peak is loaded to an HR 5/10 MonoS column (Pharmacia) and eluted in a linear gradient of 50-500 mM KCl in20 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 10% glyceroland 1 μM PMSF. The purified p68 is stored at −70 C.

[0150] B. From Recombinant E. coli Cells

[0151] Alternatively, p68 is purified from E. coil expressing human p68kinase, according to published methods (Barber et al., 1991,Biochemistry 30:10356). Briefly, E. coli strain BL21 (DE3) pLysS istransformed with a plasmid containing the coding sequence for wild-typep68 protein kinase under the control of an inducible promoter. Theresulting E. coli strain is grown to log phase, then induced to expressp68 kinase. Cells are harvested by centrifugation, and lysed bylysozyme. p68 kinase is purified from the lysate by affinitychromatography using a monoclonal antibody to p68 kinase coupled toSepharose, according to published methods (Galabru et al., 1989, Eur. J.Biochem. 178:581).

EXAMPLE 2 Preparation of Viral Inhibitors

[0152] A. VAI RNA

[0153] VAI RNA is prepared according to published methods (Mellits etal.,1990, Nucl. Acids Res. 18, 5401). Briefly, plasmid pT7VA/Ad2I,abbreviated here to pT7VA, is a derivative of the cloning vector pUC119containing the promoter for T7 RNA polymerase fused upstream of the genefor Ad2 VA RNA₂. The plasmid is linearized by digestion with Dra I toallow preparation of run-off transcripts which are exact copies of VA1RNA. Transcription is performed in reactions containing 37.5 μg/ml T7RNA polymerase, 50 μg/ml linearized pT7VA DNA, 40 mM Tris-HCl, pH 7.8,14 mM MgCl₂, 2 mM spermidine, 5 mM dithiothreitol (DTT), 4 mM each.rNTP, and 1 unit/μl RNasin (Promega) After incubation at 40° C. for 90minutes, the reaction is terminated by addition of EDTA to 20 mM,extracted with phenol and then chloroform, and the RNA is precipitatedwith ethanol. VA1 RNA is purified by denaturing the redissolvedprecipitate, running it on an 8% polyacrylamide/7 M urea sequencing gel,excising the major band, and recovering the RNA by standard methods.Labeled VA1 RNA is prepared by performing transcription as described butincluding either [alpha-³²P]UTP or biotinylated-UTP.

[0154] B. EBER-1 RNA

[0155] The EBER-1 RNA is prepared according to published methods (Clarkeet al., 1990) from the plasmid pPAC-1, which contains the T7 RNApolymerase promoter sequence upstream of the EBER-1 gene. Fortranscription of the EBER-1 RNA, plasmid pPAC-1 is linearized with Sau3AI and used as the template in an in vitro transcription reaction with T7RNA polymerase under the conditions recommended by the supplier.Following transcription, the RNA is extracted once withphenol/chloroform and once with chloroform, precipitated with ethanoland examined by electrophoresis on a non-denaturing agarose gel, toconfirm the presence of the predicted 171-nucleotide species. The EBER-1preparations are further purified by chromatography on CF11-cellulose(Whatman), to removed double-stranded RNA. Labeled EBER-1 RNA isprepared by performing transcription as described but including either[alpha-³²-P]UTP or biotinylated-UTP

[0156] C. HIV TAR RNA

[0157] HIV TAR RNA is isolated by published methods (Gunnery et al.,1990, Proc. Natl. Acad. Sci. US 87, 8687), using plasmid pEM-7, whichcontains the T7 RNA polymerase promoter bacteriophage T7 upstream of asequence corresponding to nucleotides +3 to +82 of the HIV LTR. Theplasmid is linearized by digestion with Hind III and used as a templatefor transcription of TAR RNA which is then purified essentially asdescribed in part B above. Labeled TAR RNA is prepared by performingtranscription as described but including either [alpha-³²P]UTP orbiotinylated-UTP.

EXAMPLE 3 Screening Method: Solid Phase Support for Immobilized p68Kinase

[0158] 100 ng-5 μg of a monoclonal antibody to human p68 kinase isimmobilized in each well of a microtiter plate or on nitrocellulose ineach slot of a slot-blot apparatus. After incubation for 1 hour at roomtemperature to allow antibody to bind, the plate or slot-blot is washed2-4 times with phosphate buffered saline to reduce non-specific binding.p68 kinase is then bound to the immobilized antibody as follows. 10-50μl of a cell extract containing p68 is added to each well or slot. Thep68-containing extract is either a 1:20 dilution of a cell lysate frominterferon-treated eukaryotic cells or from E. coli cells expressinghuman p68 kinase, or a partially purified preparation of p68 kinase fromeither source. After incubation for 1 hour at room temperature to allowp68 to bind, the plate or slot-blot is washed 2-4 times with phosphatebuffered saline to reduce non-specific binding. If a slot-blot apparatusis being used, the nitrocellulose sheet is now removed. Bindingreactions are performed by adding labeled VAI RNA or other viralinhibitor to each well or to the entire nitrocellulose sheet after itsremoval from the slot blot apparatus. VAI RNA is added at aconcentration of about 2-3 ng/ml in phosphate buffered saline. The plateor nitrocellulose sheet is incubated for 1 hour at room temperature, andwashed 2-4 times with phosphate buffered saline to reduce non-specificbinding. Bound VAI RNA (or other inhibitor) is quantitated byautoradiography or liquid scintillation counting for ³²P-labeled VAIRNA, or using streptavidin, biotinylated alkaline phosphatase andchemiluminescent detection for biotinylated VAI RNA. A typical testseries includes the following reactions: a) a control reaction withinhibitor but no p68 kinase; b) control reactions with test compoundalone or with either p68 kinase or inhibitor; c) a reaction includingp68 kinase and inhibitor without test compound; and (d) a reactionincluding p68 kinase, inhibitor and test compound. For test compoundswhich interfere with binding of viral inhibitor to p68 kinase, theamount of bound inhibitor detected in reaction (d) is less than thatdetected in reaction (c).

EXAMPLE 4 Slot-Blot Filter-Binding Assay

[0159] Reaction mixtures containing one or more of purified radiolabeledVAI RNA (or other viral inhibitor), purified p68 kinase, and testcompound are incubated together for 15-20 minutes on ice in the presenceof 75 mM KCl, 25 mM HEPES, (pH 7.4), 10 mM MgCl₂, 1.0 mM dithiothreitol,0.1 mM ATP, 0.1 mg/ml bovine serum albumin, 0.1 mM tRNA and 0.1 mM EDTA.Reactions are diluted with 10 volumes of wash buffer (50 mM KCl, 1.5 mMMgCl₂, 20 mM HEPES (pH 7.4), 0.1 mM EDTA), and immediately filtered in aslot-blot apparatus through a 0.45 micron pore-size nitrocellulosemembrane (Schleicher & Schuell, Keene, N.H.) that has been soaked for 1hour at room temperature in wash buffer containing 0.1 mg each of BSAand salmon sperm DNA per ml. Each well is washed with 200 μl of ice-coldwash buffer, and the filter is dried and exposed to autoradiography.Quantitation is performed by scintillation counting of individual bandsor by direct scanning of the membrane with a AMBIS Imaging System. Atypical test series includes the following reactions: a) controlreactions with p68 kinase alone or VAI RNA alone; b) control reactionswith test compound alone or with either p68 kinase or VAI RNA; c) p68kinase and VAI RNA; and d) p68 kinase, VAI RNA and test compound. Fortest compounds which interfere with binding of viral inhibitor to p68kinase, the amount of bound inhibitor detected in reaction (d) is lessthan that detected in reaction (c).

EXAMPLE 5 Screening Method: p68 Autophosphorylation Assay

[0160] In this assay, p68 kinase is incubated under kinase reactionconditions with activating double-stranded RNA, gamma-³²P ATP to followkinase autophosphorylation, VAI RNA (or other inhibitor), and a testcompound. Up to 2 μl of p68 kinase fraction (the exact volume useddepends on the degree of purification) is diluted to 10 μl with 50 mMKCl, 20 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 10%glycerol and 1 μM PMSF, 0.1 mg bovine serum albumin and 0.1 mg of tRNAper ml. The diluted kinase is added to 20-μM reaction mixturescontaining, at final concentrations, 75 mM KCl, 25 mM HEPES, (pH 7.4),10 mM MgCl₂, 1.0 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM ATP, proteaseinhibitors, and 5 to 10 μCi of [gamma ³²-P]ATP (>3,000 Ci/mmol; Dupont,NEN). Reaction mixtures are supplemented as appropriate with reovirusdouble-stranded (ds) RNA or synthetic dsRNA (e.g. poly I:C) as anactivator and VAI RNA as an inhibitor. When used in the same reaction,dsRNA and VAI RNA are added simultaneously to the enzyme mix. Thereactions are incubated at 30 C. for 15-25 min, then filtered throughnitrocellulose in a slot-blot or dot-blot apparatus, prepared as inExample 4. ³¹P incorporated into the p68 kinase by autophosphorylationis quantitated by liquid scintillation counting or by laser densitometryof an exposed autoradiographic film. A typical test series includes thefollowing reactions: a) control reactions with p68 kinase alone or VAIRNA alone; b) control reactions with test compound alone or with eitherp68 kinase or VAI RNA; c) p68 kinase and VAI RNA; and d) p68 kinase, VAIRNA and test compound. For test compounds which interfere with bindingof viral inhibitor to p68 kinase, the amount of autophosphorylated p68kinase detected in reaction (d) is more than that detected in reaction(c).

EXAMPLE 6 Preparation of p58

[0161] I. Preparation of p58 from Bovine Cells

[0162] A. General Methods

[0163] Madin-Darby bovine kidney (MDBK) cells (Etkind & Krug, 1975, J.Virology 16, 1464-1475) are grown in monolayers as described (Katze, etal., 1988, J. Virology 62, 3710). Monolayers of MDBK cells (2×10¹⁰ incells; 800 T150 flasks) are infected with influenza virus at amultiplicity of infection (m.o.i.) of 10 plaque-forming units per cellfor 4 hours. The infected cells are washed twice with ice-cold Hanks'balanced salt solution and lysed in buffer A:50 mM Tris-HCl, pH 7.5, 50mM KCl, 1 mM dithiothreitol, 2 mM MgCl₂, aprotinin at 100 μg per ml, 1mM phenylmethylsulfonyl fluoride, 1% Triton X-100. The cytoplasmicextracts are then centrifuged at 100,000×g for 1 hour in a Beckman Ti70.1 rotor. The supernatant (S100) is fractionated by ammonium sulfateprecipitation (40-60%). The ammonium sulfate precipitate is resuspendedin buffer B: 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mMphenylmethylsulfonyl fluoride, 5% glycerol supplemented with 100 mM KCland dialyzed against the identical buffer. The dialyzed sample isapplied to a Mono Q HR 10/10 column. Bound proteins are eluted with a100-ml linear gradient of 100-500 mM KCl in buffer B. Kinase-inhibitoryactivity is assayed as described in B below. The kinase inhibitorymaterial elutes at 280 mM KCl. Active fractions are pooled, concentratedby using a Centriprep 30 concentrator (Amicon, Danvers, Mass.), anddialyzed against buffer B containing 25 mM KCl. The dialyzed fraction isapplied to a heparin-agarose column and bound material is eluted bysequential application of buffer B containing, respectively, 100, 300,and 500 mM KCl. The kinase inhibitory activity is found in the 300 mMKCl fraction, which is then concentrated and dialyzed against bufferB/25 mM KCl. The dialysate is loaded onto a Mono S HR 5/5 column, andbound material is eluted with buffer B/250 mM KCl. To achieve the finalpurification, the active Mono S fraction is layered onto a 10-30%glycerol gradient containing buffer B/25 mM KCl. The gradient iscentrifuged at 49,000 rpm for 21 hours in a Beckman SW 55 rotor.Fractions are collected, dialyzed, and assayed for kinase inhibitoryactivity as described below.

[0164] B. Assay for Inhibition by p58

[0165] This assay allows purification of p58 from influenzavirus-infected cells to be monitored. Fractions isolated during the p58purification procedure are mixed with a p68-containing cell extractprepared by disruption of interferon-treated 293 cells with TritonX-100, and incubated for 20 minutes at 30 C. The p68 kinase is thenimmunoprecipitated using an antibody which recognizes the human p68 from293 cells but not the bovine homologue in influenza virus-infected MDBKcells, the source of the p58. The activity of the immunoprecipitated p68kinase is then measured using [gamma-³²P]ATP and exogenously addedhistones as substrates. To quantitate activity, histones are subjectedto polyacrylamide gel electrophoresis and excised from the gel. In thelater stages of purification, an additional assay using pure p68 kinaseand its natural substrate, eIF-2, is performed as follows. Fractionsfrom the purification are preincubated with pure p68 kinase for 10minutes at 30 C. in buffer C (17 mM Tris-HCl, pH 7.5, 75 mM KCl, 0.1 mMEDTA, 1.0 mM diethiothreitol, aprotinin at 8 μg per ml, 0.1 mMphenylmethylsulfonyl fluoride, 2 mM MgCl₂, 2 mM MnCl₂, 0.3 mg of bovineserum albumin per ml, 8% glycerol). Activator poly(I):poly(C) (0.010μg/ml) is then added in the presence of 1 mM [gamma³²P]ATP (424 Ci/mmol;1 Ci=37 GBq) and incubation continued for an additional 10 minutes.Finally, 0.5 μg of purified elF-2 is added and incubation is continuedfor a further 10 minutes at 30 C. The reaction is terminated by additionof 2×disruption buffer (160 mM Tris, pH 6.8, 1.0 M 2-mercaptoethanol, 4%SDS, 20% (vol/vol) glycerol), the mixture is boiled, and thephosphorylated proteins are analyzed on an SDS/14% polyacrylamide gel.

[0166] II. Cloning of p58

[0167] A. Screening of cDNA Library

[0168] Three tryptic peptides derived from purified p58 protein weresequenced by microsequencing. One of the sequences(AEAYLIEEMYDEAIGDYETA) was used to design a degenerate oligonucleotideprobe (5′-GAA(G)GAA(G)ATGTAT(C)GAT(C)GAA(A)GC-3′). This was used toscreen a cDNA library from the MDBK cell line made in the Lambda Zap IIvector (Stratagene). Duplicate plaque transfers were made to nylonfilters (Hybond-N; Amersham, Arlington Heights, Ill.). Filters were thenprehybridized in 6×SSPE (1×SSPE=0.18 M NaCl/0 mM NaPO₄, pH 7.4, 1 mMEDTA, 1% SDS, 0.2% Ficoll 0.2% bovine serum albumin, 0.2%polyvinylpyrrolidone), 100 μg of sonicated and denatured salmon spermDNA per ml at 38 C. for 4 hours and hybridized with ³²P-5′-end-labeledprobe in 6×SSPE, 1% SDS, 100 μg of sonicated and denatured salmon spermDNA per ml at 38° C. for 20 hours. Filters were washed in 6×SSPE, 1% SDStwice at room temperature for 10 minutes, once at 38 C. for 15 minutesand exposed at −70 C. with Kodak X-Omat film with enhancing screens.Positive phage plaques were identified and purified by further rounds ofplaque hybridization. The pBluescript plasmid (Stratagene) was excisedout in vivo according to the manufacturer's instructions. EcoR Ifragments from 4 positive clones were analyzed by Southern blothybridization using the degenerate oligonucleotide probe p58-3-2 (5′-GCIGTT(C)TCA(G)TAA(G)TCT(C)TG-3′; I represents inosine) correspondingto the antisense-strand of a partial amino acid sequence (QDYETA) of thep58. one positive clone containing an insert of 1400 bp was obtained andanalyzed by restriction enzyme mapping. After cloning into M13mp18 andM13mp19, the sequence of the p58 cDNA was determined by thedideoxynucleotide chain-termination method using Sequenase 2.0 (UnitedStates Biochemical). See SEQ ID No. 17. Sequence data were analyzedusing the Genetics Computer Group (GCG) sequence-analysis program(version 7.0).

[0169] B. Isolation of the 3′ End Region of p58 cDNA

[0170] The initial clone isolated contained a long open-reading framebut no termination codon, suggesting that the 3′-end was missing. Themissing 3′ end region was isolated using RACE-PCR (Rapid Amplificationof cDNA ends-polymerase chain reaction) as described (Innis, et al.,1990). MDBK poly (A)+mRNA (1 μg) was reverse-transcribed using a hybridprimer (5′-GACTCGAGGATCCGAATTC-(T)₁₇-3′). The cDNA pool was amplified byRACE-PCR in the presence of adapter primer (5′-GACGCGACCATCCGAATTC-3′)and p58 gene-specific primer P58-5 (5′ GCTGAAGAGCTCATCAAAG-3′) under theconditions as described (Innis, et al., 1990). After identifying theamplified product by Southern blot, the product was isolated from anagarose gel and cloned into M13mp18 and m13mp19 to sequence theamplified region. This allowed reconstruction of the complete p58 cDNAcontaining 1680 bp. The original 1400 bp cDNA was also used to screenthe MDBK cDNA library and pull out another clone of 3140 bp containingthe full coding sequence together with 5′-and 3′-UTRs.

[0171] C. Expression of Fusion Protein in Bacteria

[0172] A unique Nde I site (CATATG) was introduced at the initiatingmethionine codon of the p58 gene using an in vitro mutagenesis kit(Bio-Rad, Richmond, Calif.) according to the manufacturer's protocol.After site-directed mutagenesis, a 1.6 kb Nde 1-BamH I fragmentcontaining the p58 gene was cloned into the bacterial expression vectorpET15b (Novagen, Madison, Wis.). p58 was expressed as a histidine-taggedfusion protein in E. coli BL21 (DE3)pLysS after inducing with 0.2 mMIPTG for 2 hours at 30° C. Most of the fusion protein was found in theinsoluble fraction. After denaturing this fraction in 6 M Guanidium-HCl,the fusion protein was purified using a Ni(II)-column in accordance withthe manufacturer's instructions. The purified protein (0.1 mg/ml) wasrenatured after diluting about 50-fold in the dialysis buffer (20 mMTris-HCl, pH 7.5, 1 mM DTT, 0.1 mM EDTA, 0.15 M NaCl, 20% glycerol)containing 0.1 mg bovine serum albumin per ml and dialyzing in dialysisbuffer at 4° C. for 6 hours. The renatured protein was aliquoted andstored at −70° C.

EXAMPLE 7 Screening Method: Inactivation of p68 by p58

[0173] This assay is performed in essentially the same way as theprocedure in example 5. Up to 2 μl of p68 kinase fraction (the exactvolume used depends on the degree of purification) is diluted to 10 μlwith 50 mM KCl, 20 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 0.1 mM EDTA, 1 mMDTT, 10% glycerol and 1 μM PMSF, 0.1 mg bovine serum albumin and 0.1 mgof tRNA per ml. The diluted kinase is added to 20-μl reaction mixturescontaining, at final concentrations, 75 mM KCl, 25 mM HEPES, (pH 7.4),10 mM MgCl₂, 1.0 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM ATP, proteaseinhibitors, and 5 to 10 μCi of [gamma ³²P]ATP (>3,000 Ci/mmol; Dupont,NEN). Reaction mixtures are supplemented as appropriate with reovirusdouble-stranded (ds) RNA or synthetic dsRNA (e.g. poly I:C) as anactivator and p58 as inhibitor. When used in the same reaction, dsRNAand p58 are added simultaneously to the enzyme mix. The reactions areincubated at 30 C. for 15-25 min, then filtered through nitrocellulosein a slot-blot or dot-blot apparatus, prepared as in Example 4. ³²Pincorporated into the p68 kinase by autophosphorylation is quantitatedby liquid scintillation counting or by laser densitometry of an exposedautoradiographic film; A typical test series includes the followingreactions: a) control reactions with p68 kinase alone or p58 alone; b)control reactions with test compound alone or with either p68 kinase orp58; c) p68 kinase and p58; and d) p68 kinase, p58 and test compound.For test compounds which interfere with binding of p58 inhibitor to p68kinase, the amount of autophosphorylated p68 kinase detected in reaction(d) is more than that detected in reaction (c).

EXAMPLE 8 In vitro Translation Assay

[0174] The following components are added in sequence to 12 μl ofmicrococcal nuclease-treated rabbit reticulocyte lysate: 2.5 μl of 50mCi/ml [³⁵S] methionine, 2.5 μl of an amino acid mixture containing 1 mMof all amino acids except methionine, 2.5 μl of 50 μg/ml VAI RNA, 2.5 μlof 100 ng/ml reovirus double-stranded RNA, and 2 μl of test compound orH₂O. The mixture is incubated at 30 C. for 15-20 min to allow activationof endogenous p68 kinase, then 1 μl of specific reporter gene mRNA isadded to give a final concentration of 10 μl/ml. The translationreaction is then incubated for 30 min at 30 C. Translation isquantitated by SDS-PAGE and autoradiography, by CAT or luciferase enzymeassays, or other assay as appropriate for the mRNA used. A typical testseries includes the following reactions: a) control reaction withoutreovirus dsRNA or VAI RNA; b) control reaction with reovirus dsRNA butwithout VAI RNA; c) reovirus dsRNA and VAI RNA; and d) reovirus dsRNA,VAI RNA, and test compound. For test compounds which interfere with VAIRNA function, translation in reaction (d) is reduced compared to thatdetected in reaction (c).

EXAMPLE 8a Identifying Antisense Oligodeoxynucleotide Molecules thatInterfere with the Function of an Adenovirus Gene Product, VAI RNA

[0175] The following example provides compositions and methods whichspecifically block the function of adenovirus VAI RNA, which is known toinhibit the activation of a cellular antiviral enzyme p68. Inhibition ofp68 activation is important for replication of adenovirus in vivo, andessential for viral resistance to interferon. Any compound thatspecifically blocks the function of VAI RNA would be expected to inhibitadenovirus replication since it would inhibit a key step in the virallife cycle.

[0176] The interferon response is a primary defense mechanism againstviruses. In response to viral infection, mammalian cells secreteinterferon, which in turn induces the production of several enzymes thathave antiviral effects (Sen, G. C. and P. Lengyel. 1992, J. Bio. Chem.267:5017-5020). One of these enzymes is a protein kinase designated hereas p68, but also known as PKR, for Protein Kinase RNA-activated, eIF2kinase, dsRNA-PK, DAI, P1 kinase, and dsI. This enzyme is induced in aninactive form by interferon, and is activated only after interactionwith double stranded RNA (dsRNA), which is usually produced during viralinfection (Hershey, J. W. B. 1993, Seminars in Virology 4:201-207.Samuel, C. E. 1993, The eIF-2a protein kinases, regulators oftranslation in eukaryotes from yeasts to humans. J. Bio. Chem.268:7603-7606). Once activated, p68 phosphorylates eukaryotic initiationfactor 2 (eIF2). Phosphorylation of eIF2 leads to inhibition oftranslation, both cellular and viral. Since viruses are obligateintracellular parasites that depend on their host cell for translation,the viral life cycle is blocked by this inhibition of translation.

[0177] Many viruses possess counterdefenses to allow them to replicatein spite of interferon and its induced antiviral enzymes. Severalviruses are known to produce specific inhibitors of p68 (Katze, M. G.1993, Seminars in Virology 4:259-268. Mathews, M. B. 1993, Seminars inVirology 4:247-257). Among these is adenovirus, which produces aspecialized RNA, designated VAI RNA, that specifically inhibits p68. VAIRNA is essential for the observed resistance of adenovirus tointerferon; mutants without a functional VAI RNA are sensitive tointerferon. VAI RNA is a 150 base single stranded RNA molecule withinternal base pairing that results in a complex structure of doublestranded stems and single stranded loops (FIG. 1). The molecule isroughly divided into regions designated the terminal stem, the centraldomain, and the apical stem-loop. Some of these regions of secondarystructure have been identified as essential to the function of VAI RNA(Mathews, M. B. and T. Shenk. 1991, J. Virol. 65:5657-5662. Mellits, K.H., T. Pe'ery, and M. B. Mathews 1992, J. Virol. 66:2369-2377).

[0178] Miroshnichenko, et al, 1989. “Inhibition of adenovirus 5replication in COS-1 cells by antisense RNAs against the viral E1Aregion.” Gene 84:83-89 reported that antisense RNA to the earlyadenovirus gene product E1A could reduce plaque yield. E1A isfunctionally unrelated to VAI RNA, and does not play a role ininhibition of p68. Their experiments did not use exogenously addedantisense oligodeoxynucleotides, but relied on the transfection of aplasmid encoding an antisense RNA.

[0179] Based on the known base sequence of VAI RNA, the predictedsecondary structure (FIG. 1), and the relative importance of the variousstems and loops to VA function, applicant designed several antisenseoligodeoxynucleotide molecules (FIGS. 2,3). These antisense-species weretested in the in vitro translation assay described above.

[0180] A. In vitro Translation Assay

[0181] The in vitro translation assay was performed in a 96 well plate.Rabbit reticulocyte lysate was used as a source of p68 as well as an invitro translation system. p68 is present in rabbit reticulocyte lysates,and can be activated by the addition of dsRNA such as reovirus RNA. Whenp68 is activated, translation is inhibited. Means for monitoring levelsof translation are utilized. For example, translation can easily bemonitored by assaying for reporter gene expression, such aschloramphenicol acetyltransferase and others as are known in the art. Inone embodiment luciferase may be used as a reporter protein since it canbe quantitated in a luminometer. Compounds that activate p68 (e.g.reovirus RNA) will cause inhibition of translation and result in adecreased reporter protein, such as luciferase, signal. Addition of VAIRNA to the reaction containing reovirus RNA will result in an increasein luciferase signal as the VAI RNA inhibits activation of p68 andallows translation to continue. Antisense oligodeoxynucleotides wereadded to the reaction containing reovirus RNA and VAI RNA. Antisensespecies that interfere with VA1 RNA function will lead to a decrease inluciferase signal.

[0182] VAI RNA was prepared as described in the main body of this patent(see Example 2: Preparation of viral inhibitors, A. VAI RNA). Thefollowing components were added in order and incubated as indicated inwells of a 96 well plate:

[0183] 4 μl of antisense oligodeoxynucleotide (final concentration is 20fold molar excess to VAI RNA)

[0184] 2 μl of VAI RNA (final concentration 5 μl/ml)

[0185] 20 μl rabbit reticulocyte lysate supplemented with 1 mM completeamino acid mixture.

[0186] * Incubate at 30° C. for 15 min.

[0187] 2 μl reovirus dsRNA (final concentration=10 ng/ml)

[0188] * Incubate at 30° C. for 15 min.

[0189] 2 μl luciferase mRNA (final concentration: 30 ng/ml)

[0190] * Incubate at 30° C. for 15 min.

[0191] The wells were immediately assayed for luciferase activity usinga Dynatech ML3000 luminometer and Analytical Luminescence Labs EnhancedLuciferase Assay Kit. Settings were: Enhanced Flash mode, Delay=2s,Integrate=5s, 50 μl Substrate A injected simultaneously with 50 μlSubstrate B.

[0192] The following controls were included in the assay. H₂O was alsosubstituted for mRNA, reovirus RNA, cr VAI RNA as indicated by (−).

[0193] 1) (−) mRNA (−) reo (−) VA

[0194] 2) (+) mRNA (−) reo (−) VA

[0195] 3) (+) mRNA (+) reo (−) VA

[0196] 4) (+) mRNA (+) reo (+) VA

[0197] Control 1 tested for the presence of anything in the lysate thatmight give a positive luciferase signal without luciferase mRNA. Control2 established normal level of translation for the assay. Control 3established the level of inhibition of translation that was a result ofreovirus RNA activation of p68. Control 4 determined the rescue functionof VAI RNA as an inhibitor of p68 in the presence of the activator,reovirus RNA. Typical results are shown in FIG. 4. The production ofRelative Light Units (“R.L.U.”) is dependent on the addition ofluciferase mRNA (column 1 vs. 2). When reovirus dsRNA was added to theassay, the endogenous p68 in the lysate was activated and translationwas inhibited (column 3). When VAI RNA was added, translation waspartially rescued (column 4).

[0198] B. Antisense Oligodeoxynucleotide Results

[0199] Antisense oligodeoxynucleotides species were added to the assay;these results are also shown in FIG. 4. Ava 1 was found to completelyreverse the effect of VAI RNA (column 5). Ava 9, and ava 15 werepartially antagonistic to VAI RNA function (columns 8, 9). Ava 2 and 3did not significantly affect VAI RNA function (columns 6, 7). Otherantisense species, including species complementary to the terminal stemregion, did not interfere with VAI RNA function (data not shown). Theselatter results underscore the specificity of inhibition by ava 1, sinceantisense to some parts of the VAI RNA molecule are not effective inblocking function.

[0200] Applicant shows that the aforesaid block of VAI RNA function isnot dependent on RNase H cleavage of the RNA-DNA hybrid formed by VAIRNA and ava species. Therefore, the sequences of theoligodeoxynucleotides designated ava 1, 9, and 15 synthesized asmodified RNA or DNA would be expected to function effectively. By“modified DNA or RNA” is meant that nucleic acid base analogs as areknown in the art may be present, for example, DNA analogs could include,but are not limited to, methylphosphonate DNA or phosphorothioate DNA.DNA analogs may provide advantages such as nuclease resistance andincreased cellular uptake. Additionally, one base, for example adeninemay be substituted for another base, for example, guanine; thephosphodiester linkage may be modified as is known in the art, forexample by substitution of a thioester linkage; or the sugar moiety ofthe nucleic acid may be modified as is known in the art, for example,substitution of 2′-deoxyribose with ribose or substitution of ribosewith 2′-deoxyribose. These modifications may be made to one or morebases in the nucleic acid sequence. Modifications also include changeswhich, for example, stabilize the nucleic acid, but do not effect thefunction of the nucleic acid (as can be determined by routine testing).

[0201] Not every antisense oligodeoxynucleotide interferes with VAIfunction, as shown by the fact that ava 2 and several other antisensespecies complementary to various regions of VAI RNA did not affect VAIactivity. Ava 2 is complementary to part of the central domain of VAIRNA. Since this complementary region of VAI RNA is single-stranded, ava2 would be expected to anneal readily, and since the central domain hasbeen shown to be critical for function (Mathews, M. B. and T. Shenk.1991. Adenovirus virus associated RNA and translation control. J. Virol.65:5657-5562. Mellits, K. H., T. Peery, and M. B. Mathews. 1992. Role ofthe apical stem in maintaining the structure and function of adenovirusvirus associated RNA. J. Virol. 66:2369-2377), ava 2 would be expectedto interfere with VAI function. However, ava 2 did not affect VAIactivity (FIG. 4).

[0202] In contrast, ava 15 is complementary to a double-stranded regionof VAI RNA. This region would not be expected to easily allow binding ofan oligodeoxynucleotide, and yet the antisense oligodeoxynucleotide ava15 does in fact antagonize VAI function (FIG. 4). By utilizingApplicant's method, screening of oligonucleotides for those which arefunctional may be easily accomplished. More generally, the readydetection by the Applicant's screening method of the unexpected natureof the behavior of both ava 2 and ava 15 demonstrates the utility of themethod for identifying antagonists of viral inhibitors of p68.

EXAMPLE 9 Monitoring p68 Activity as a Function of Translation ofGCN4-reporter Gene Fusions in in vitro Extracts

[0203] To provide a positive signal in response to activation of p68kinase, in vitro translations are performed using an mRNA which carriespart of the untranslated leader for the yeast GCN4 protein fused to thecoding sequence for beta-galactosidase. Translation of such GCN4 fusionsin yeast cells is increased in the presence of activated p68 kinase.

[0204] Plasmids pM23 and pM226 (Miller & Hinnebusch, 1989, Genes Dev. 3,1217) each carry a GCN4-lacZ fusion and genes necessary for plasmidselection and maintenance in E. coli and S. cerevisiae. These twoplasmids differ by a single nucleotide: whereas pM23 has the twoupstream open reading frames (ORF1 and ORF4) which together conferp68-sensitive regulation, mutation of the ORF1 ATG codon leaves pM226with only ORF4 which by itself confers constitutive, low levelexpression. In order to provide a T7 promoter for efficient in vitrotranscription, the Sal I-Bgl II fragments of pM23 and pM226 are replacedwith a PCR-generated fragment (PCR-1 or PCR-2, respectively) as follows.PCR-1 and PCR-2 are made using oligos T7-1 (5 gcg tcg act aat acg actcac tat agg gag TCT TAT ATA ATA GAT ATA CAA AAC, with lower case for aSal I recognition site and the T7 RNA polymerase promoter, and uppercase for GCN4 sequence starting with the 5′ end of the native mRNA), andT7-2 (5′ GGG AAA TTT TTA TTG GCG AGT AAA CCT GG, residues 503 to 475,relative to the transcription start site) as primers, plasmids pM23 andpM226, respectively, as templates, and a standard GeneAmp™ PCR kit fromPerkin Elmer. The PCR-generated fragments are cloned directly using theTA-cloning kit from Invitrogen. The promoter fragments are excised withSal I and Bgl II and subcloned into pM23 and pM226, respectively, thathave been digested with the same two enzymes.

[0205] The modified plasmids are transcribed in vitro with T7 RNApolymerase and translated in vitro as described above.Beta-galactosidase activity is measured using standard assay conditionsfor the enzyme. If it becomes desirable to use a reporter gene otherthan beta-galactosidase, the lacZ gene is bracketed by two Bam HI sites,which can be used for excision and replacement with the new reportergene.

EXAMPLE 10 Construction of Yeast Strain for Screening

[0206] The starting point for the p68 kinase assay strain is the straindesignated H1895, which has the genotype: a ura3-52 leu2-3 leu2-112trp1-Δ63 gcn2Δ[GCN4-lacZ TRP1] at trp1-Δ63 (Dever et al., 1993, Proc.Natl. Acad. Sci. US). Because this strain is deleted for GCN2, it lacksthe kinase normally responsible for inducing the GCN4 pathway and istherefore dependent upon an exogenous kinase (i.e., the mammalian p68kinase) for activating GCN4. Expression of GCN4 is convenientlymonitored in this strain by using a GCN4-lacZ fusion which directs thesynthesis of beta-galactosidase under GCN4 control. The plasmids p1420and p1419 (Id.) are, respectively, high and low copy number URA3plasmids, which contain the cloned p68 kinase gene under the control ofthe GAL-CYC promoter.

[0207] Plasmid pMHVA (Mellits & Mathews, 1988, EMBO J., 7 2849-2859)containing the gene encoding VAI RNA was digested with Xba I and Pst Iand the fragment containing the VAI RNA gene, its promoter andtranscription terminator was inserted into high- and low-copy numberLEU2 plasmids p425 & p315, respectively (Sikorski & Heiter, Genetics,122. 19-27, 1989; Christianson et al., Genetics, 110, 119-122, 1992)that had been digested with Spe I and Pst I. All restriction digestionsand ligations were performed according to manufacturers′ instructions.

[0208] Strain H1895 was then transformed with all pairwise combinationsof the low and high copy number plasmids containing the VAI RNA and p68kinase genes by selecting for growth on minimal medium lacking histidineand leucine. This yielded a battery of strains suitable for evaluatingthe interaction between p68 and VAI RNA (in the presence or absence ofgalactose) and for choosing which combination of plasmids is optimum forthe desired assay.

EXAMPLE 11 In vitro Assays for Degradation of p68 by Poliovirus

[0209] To prepare radiolabeled p68, suspension HeLa cells are incubatedin medium containing [³⁵S]methionine (500 μCi/ml) together with humanlymphoblastoid alpha and beta interferon for 16 hours. After harvest,cells are washed in ice-cold Hank's Balanced Salt Solution (HBSS) anddisrupted in lysis buffer (10 mM Tris.HCl, pH 7.5, 50 mM KCl, 2 mMMgCl₂, 2 mM MnCl₂, 0.1 mM EDTA, 0.2 mN phenylmethylsulfonyl fluoride, 1%Triton X-100). Alternatively, radiolabeled p68 is prepared by in vitrotranscription of a cDNA clone followed by in vitro translation in wheatgerm extracts under standard conditions and in the presence of[³⁵S]methionine.

[0210] To prepare test extracts from infected and uninfected cells, HeLacells are grown in suspension and either infected with poliovirus ormock infected five hours before harvest. Cells are then harvested andextracts made as for radiolabeled cells. To test for p68-degradingactivity, the extract from radiolabeled cells is mixed with extract fromeither infected or mock-infected cells and incubated at 30° C. for 15minutes. Alternatively, the radiolabeled products of the in vitrotranscription and translation of the p68 cDNA clone are mixed withextract from either infected or mock-infected cells and incubated at 30°C. for 15 minutes. In either case, radiolabeled p68 isimmunoprecipitated after incubation with the cell extracts using amonoclonal antibody (available from A. Hovanessian, Institut Pasteur,although other antibodies are readily prepared with equivalent effect)bound to Sepharose. Immunoprecipitated material is subjected topolyacrylamide gel electrophoresis, detected by autoradiography andquantitated by laser densitometry.

EXAMPLE 12 Partial Purification of p68 Proteolytic Activity fromPoliovirus-infected Cell Extracts

[0211] 2×10⁴ HeLa cells grown in suspension are infected with poliovirusfor 5 hours at a multiplicity of infection (m.o.i.) of 20 plaque-formingunits (pfu) per cell. As a control, a similar number of cells aremock-infected for the same period. Cells are harvested and washed inice-cold Hank's Balanced Salt Solution (HBSS) and then disrupted inlysis buffer (10 mM Tris.HCl, pH 7.5, 50 mM KCl, 2 mM MgCl₂, 2 mM MnCl₂,0.1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100).Nuclei and membranes are removed by centrifugation at 4,000×g for 10minutes. Pooled extracts from infected or mock-infected cells aresubjected to sequential differential precipitations using ammoniumsulfate at 20%, then 40%, then 60%, and finally at 80% saturation.Pellets are resuspended and dialyzed against lysis buffer containing 5%glycerol. The pellet and supernatant from each precipitation is testedin the p68 degradation assay described above (Example 9).

[0212] Nucleic Acid Targets

[0213] One particularly useful macromolecule target is a nucleic acid.There now follows a detailed review of useful methods of this inventionwhich are based upon targeting agents of this invention to such nucleicacids.

[0214] Viruses are believed by Applicant to employ nucleic acidsequences responsible for preferential translation of viral RNAs.Viruses whose RNAs are believed to be preferentially translated becauseof specific viral nucleic acid sequences currently includepicornaviruses, hepatitis B virus, hepatitis C virus, influenza virus,adenovirus and cytomegalovirus.

[0215] Picornaviruses are an important class of viruses responsible fora broad array of human and animal diseases (reviewed in Chapters 20-23in Fields B N, Knipe D M (eds): Fields Virology, ed. 2, Raven Press, NewYork, 1990). They include polioviruses, rhinoviruses (the most frequentcause of respiratory tract infections), coxsackie viruses (a cause ofgastrointestinal illnesses, myocarditis and meningitis), hepatitis Avirus, and foot-and-mouth disease viruses. Picornaviruses aresingle-stranded RNA viruses whose RNA genomes are positive-sense andnonsegmented. The genomic RNA strand inside each virus is translatedwhen the virus enters a host cell. One of the proteins translated fromthe incoming RNA genome is an RNA-dependent RNA polymerase which copiesthe viral genome to produce additional full-length viral RNAs. Some ofthese RNAs are translated to produce additional viral proteins, and someare packaged as RNA genomes into a new generation of viruses. Each RNAis translated into a single “polyprotein” which is cleaved as it istranslated to yield individual viral proteins.

[0216] One of the early effects of infection with a picornavirus is ashutoff of host protein synthesis. At least in the case of poliovirusinfection, this appears to be due to cleavage of a host cell proteinknown as p220, one of three polypeptide constituents of the initiationfactor eIF-4F, also known as cap-binding protein complex. eIF-4F isrequired for initiation of protein synthesis from host cell mRNAs, whichbear a structure known as a cap at their 5′-ends. eIF-4F is believed tobind to the cap structure and participate in the unwinding of secondarystructure adjacent to the cap in the 5′-untranslated leader (5′-UTR) ofmRNAs. This unwinding is necessary for ribosomes to bind to the mRNA andmigrate along it to the AUG codon which represents the start of thecoding sequence. Thus, by cleaving one of the subunits of eIF-4F,picornaviruses prevent cap-dependent initiation of translation ofhost-cell mRNAs, and thereby disable host-cell protein synthesis. ViralRNAs can be translated, however, because they utilize a cap-independentmechanism for initiation; indeed, picornaviral RNAs do not have caps attheir 5′-ends. Some but not all scientists in the field believe that thecap-independent mechanism involves sequences within the 5′-UTR of theviral RNAs known as internal ribosomal entry sites (IRES, or IRESelements) or ribosomal landing pads (RLPs) (reviewed in Sonenberg &Meerovitch, 1990). As their names imply, these are sequences whichenable ribosomes to bind to viral RNAs at internal sites rather than atthe 5′-ends of these RNAs; having bound, the ribosomes can then migrateto the AUG initiator codon and begin translation. Such binding atinternal sites allows the ribosomes to bypass the virus-induced defectin the normal cap-dependent mechanism of initiation.

[0217] The existence of IRES elements in picornaviral RNAs was inferredfrom several different types of observation (see Sonenberg & Meerovitch,1990). So, for example, viruses with mutations in the 5′-UTR were foundto make significant amounts of viral RNA but very little viral protein.More direct evidence came from the studies with dicistronic mRNAs inwhich the poliovirus 5′-UTR (for example) was positioned between thecoding sequences for two separate proteins in a single mRNA. Experimentsboth in vivo and in vitro demonstrated that the second cistron could betranslated under conditions in which the first was not, for example, invirus-infected cells or in the presence of an inhibitor of cap-dependenttranslation, but that in the absence of the viral 5′-UTR from theintercistronic space, translation of the second cistron depended ontranslation from the first. Further refinement of such experiments,involving for example progressive deletions from either end of the5′-UTR, permitted more precise definition of the region within the5′-UTR which constitutes the IRES element. Proteins which interact withIRES elements were then identified by gel-retardation assays andUV-cross-linking studies.

[0218] Evidence that IRES elements are indeed important for translationhas been obtained by demonstrating that the 5′-UTR ofencephalomyocarditis virus (EMCV) or fragments thereof can act ascompetitive inhibitors of translation in vitro (Pestova et al. (1991) J.Virol, 6194-6204) and that short DNAs complementary to the EMCV IRESelement can also block translation in vitro. (Shih et al., (1987) J.Virol. 2033-2037, Pestova et al. (1989) Virus Research, 107-118Borovjagin et al., (1991) Nucl. Acids Res., 4999-5005).

[0219] Despite these studies there is still controversy about whethertranslational initiation at IRES elements really occurs, and someevidence to suggest that it does not. Thus, one authority in the fieldhas argued strongly that important controls were omitted from crucialexperiments supporting the existence of IRES elements, characterizingthese experiments as flawed or inconclusive and IRES elements asartifacts (Kozak (1989) J. Cell Biol. 229-241; Kozak (1992) Crit. Rev.Biochem. Mol. Biol. 385-402). It has also been demonstrated that if acap is added to poliovirus RNA, which does not normally have such astructure, translation of the poliovirus RNA is inhibited (Hambridge S J& Sarnow P, (1991) J. Virology 65, 6312-6315). This observation is atodds with the purported ability of ribosomes to initiate translation ofpoliovirus RNA by binding to IRES elements downstream of the 5′-cap.

[0220] Even if IRES elements do function as their proponents claim, themechanism may not be unique to viruses. Thus it has been reported thatinternal ribosome entry sites exist within cellular mRNAs (Macejak &Sarnow (1991) Nature, 90-94; Jackson (1991) Nature, 14-15). Theexistence of such sites within cellular mRNAs would suggest that it maybe difficult to identify compounds which prevent translationalinitiation at viral IRES elements without adversely affecting thetranslation of at least some cellular mRNAs.

[0221] Picornaviruses may not be the only viruses which utilize specialsequences to enable ribosomes to bind at internal sites within RNAs andthus ensure preferential translation of viral proteins. Evidence for asimilar mechanism has also been found in the case of hepatitis B virusand hepatitis C virus. Note that since hepatitis A virus is apicornavirus, this means that virtually all clinically significanthepatitis disease is caused by viruses which utilize internal ribosomeentry sites.

[0222] Hepatitis B virus is a hepatovirus which can cause severe liverdisease and which is very widespread (reviewed in chapter 78 of Fields BN, Knipe D M (eds) Fields Virology, ed. 2, Raven Press, New York, 1990).The virus has a very unusual genome and an equally unusual method ofreplication. In brief, the viral genome consists of partiallydouble-stranded DNA. The negative-sense strand is a full circle, but thetwo ends of this circle are not covalently joined. The positive-sensestrand is incomplete and its length is not the same in all molecules, sothat the single-stranded region of the genome varies in length fromapproximately 15%-60% of the circle length in different molecules. Whenthe virus infects a cell, the infecting genome appears to be convertedto closed circular (cc) viral DNA which can be detected in the cellnucleus. This DNA is transcribed into (positive-sense) viral mRNAs, oneof which encodes a reverse transcriptase which makes negative-sense DNAcopies of viral RNA to produce further viral genomes. The (incomplete)positive-sense DNA strand of the genome is produced by partial copyingof the negative-sense strand, with synthesis primed by a short viraloligoribonucleotide. The viral reverse transcriptase (P protein) isencoded within a long mRNA which also includes the coding sequence forthe major viral core protein (C protein). The C-protein sequence isupstream of the P-protein sequence in the mRNA and partially overlapsit, in a different reading frame. Data from gene fusions which place areporter gene downstream of the C-P overlap region suggest thattranslation of the P protein involves initiation at an internal ribosomeentry site within the C-protein coding sequence (Chang et al., (1990),Proc. Natl. Acad. Sci. USA 87, 5158-5162). This interpretation issupported by the observation that defined fragments of the C-proteinsequence increase translation of the downstream cistron when placedbetween the two cistrons of a dicistronic mRNA or in the 5′-UTR of amonocistronic mRNA (Jean-Jean et al., (1989) J. Virology 63, 5451-5454).Thus, the ability to translate a crucial viral protein is highlydependent upon the presence of a specific viral nucleic acid sequencetranslationally linked to the coding sequence.

[0223] Hepatitis C virus also appears to utilize specific viral nucleicacid sequences to bypass the normal cellular method for initiation oftranslation. As its name implies, hepatitis C is a causative agent ofthe diseases formerly known as non-A, non-B hepatitis. Likepicornaviruses it has a positive-sense, single-strand genome which istranslated as a single open-reading frame, presumably into a polyproteinprecursor which is then cleaved to provide mature viral proteins. Giventhe much more recent discovery of hepatitis C virus, much less is knownabout it than the picornaviruses, and the evidence supporting its use ofIRES-like elements is unclear. Thus on the one hand, experiments basedon in vitro translation reactions led to the conclusion that translationof viral RNAs can be initiated at internal ribosome entry sites, but onthe other hand, experiments in vivo found no evidence for such amechanism of initiation (Yoo et al. (1992) Virology 889-899).

[0224] Influenza viruses also cause a dramatic inhibition of host cellprotein synthesis during infection, while viral proteins are synthesizedselectively and efficiently. Influenza viruses are of course theetiologic agents of the eponymous disease (for a review of these virusessee chapters 39 & 40 of Fields BN, Knipe DM (eds): Fields Virology, ed.2, Raven Press, New York, 1990). They too have single-stranded RNAgenomes, but in their case the genome consists of negative-sense RNA andeach gene exists on a separate RNA segment which is encapsidatedseparately into the virion; the viruses are thus of the type knowncollectively as -segmented negative-strand RNA viruses. After infectionthe separate RNAs are copied into positive-sense RNAs for translation.This copying is catalyzed by a virus-coded RNA-dependent RNA polymeraseprotein, but requires short capped pieces from the 5′-ends of cellularmRNAs to act as primers. These primers are derived from cellular mRNAsthrough the action of a virus-encoded endoribonuclease. Thus, the first10-13 nucleotides of each positive-sense, translatable, influenza viralRNA is derived from cellular mRNA.

[0225] In cells infected with an influenza virus, newly synthesizedcellular mRNAs do not reach the cytoplasm (Katze & Krug, (1984) Mol.Cell. Biol. 4, 2198-2206), and translation of pre-existing mRNAs isblocked at both the initiation and elongation stages (Katze etal.,(1986) J. Virology 60, 1027). Evidence that specific RNA sequencesin influenza virus mRNA ensure its preferential translation came fromthe fact that influenza mRNAs were selectively translated in cellsinfected by another virus, adenovirus, despite the shutdown of hostprotein synthesis in these cells (Katze et al. 1986). Further progressin understanding the preferential translation of influenza RNAs camewith the development of a transfection-infection assay (Garfinkel &Katze, (1992) J. Biol. Chem. 267, 9383-9390). This was used to show thatan exogenously introduced influenza viral gene was not subjected to thesame translational blocks in infected cells as an exogenously introducedcellular gene. It was also concluded that translation of influenza mRNAsoccurs in a cap-dependent manner, because such translation was inhibitedby poliovirus infection, which blocks cap-dependent translation. Giventhat the 5′-ends of viral mRNAs are capped and derived from cellularmRNAs, this is not unexpected. For the same reason, it would not beexpected that the 5′-UTR would play an important role in thepreferential translation of influenza mRNA. Indeed, it was observed thatthere is nothing remarkable about the primary/secondary structure orlength of the influenza 5′-UTR used for the transfection-infectionassays described above. Unexpectedly, however, it has now beendemonstrated that preferential translation of influenza mRNAs doesdepend on the 5′-UTR, and that the selectivity-determining region issurprisingly small, as small as 12 nucleotides. For comparison, atypical IRES element in a picornavirus has a length of about 400nucleotides.

[0226] Most of the viruses so far described have been RNA viruses, butDNA viruses also appear to utilize special nucleic acid sequences whichmediate preferential translation of viral RNAs. Adenovirus is an exampleof such a DNA virus (reviewed in chapters 60 & 61 of Fields BN, Knipe DM(eds): Fields Virology, ed. 2, Raven Press, New York, 1990). Adenovirusis responsible for various disorders including respiratory tractinfections, conjunctivitis, hemorrhagic cystitis and gastroenteritis.The replicative cycle of adenovirus is significantly more complicatedthan that of the smaller picornaviruses and influenza viruses. ViralRNAs are transcribed from viral DNA by the host RNA polymerase II in twomain phases, early and late transcription, with the late stage bydefinition starting with the onset of viral DNA synthesis, which isusually 6-9 hours after infection. That there is preferentialtranslation of viral RNAs is demonstrated by a variety of observations.Host-cell protein synthesis is dramatically reduced in infected cells,even though cellular mRMA synthesis continues and there is no rapidbreakdown of existing cellular mRNAs. Early in infection, early viralmRNA constitutes less than 0.1% of the total mRNA in the cell, but 5-18%of the mRNA in polysomes, that is, 5-18% of the mRNA which is beingactively translated.

[0227] The mechanisms by which adenovirus accomplishes its takeover ofprotein synthesis are not fully understood, but it has been demonstratedthat dephosphorylation of a component of the cap-binding proteincomplex, eIF-4E, may play a role in this takeover (Huang & Schneider,(1991), Cell 65, 271-280). In support of this, it has also been shownthat adenovirus mRNAs containing special sequences known as tripartiteleader sequences are translated in a cap-independent manner (Dolph etal., (1988) J. Virology 62, 2059-2066). Thus, preferential translationof adenovirus mRNAs also appears to depend upon specific viral nucleicacid sequences.

[0228] A DNA virus belonging to the herpes family, cytomegalovirus, mayalso utilize specific viral nucleic acid sequences to ensurepreferential translation of viral RNAs. Cytomegalovirus is endemic inmany populations, but many infections are subclinical in normal healthyindividuals (reviewed in chapter 69 of Fields B N, Knipe D M (eds):Fields Virology, ed. 2, Raven Press, New York, 1990). The virus cancause serious illness, however, in immunosuppressed individuals, and hasbecome a significant pathogen in recent years as a result of the rapidgrowth in the number of such individuals, some of them transplantrecipients on immunosuppressive regimens, many of them sufferers fromAIDS.

[0229] As viruses go, cytomegalovirus has a very large genome, and itsreplicative cycle and interactions with host cells are complex. Severalobservations suggest an important role for translational control of theproduction of important viral proteins (Geballe A P & Mocarski E S(1988), J. Virology.62, 3334-3340; Biegalke B & Geballe A P (1990)Virology 177, 657-667; Schleiss et al., (1991), J. Virology 65,6782-6789). Thus, several cytomegalovirus proteins, including theglycoprotein gp48, are not synthesized efficiently until late ininfection, although their mRNAs accumulate at earlier stages. Furtherinvestigations revealed an unusual cis-acting sequence in the 5′-UTR ofgp48 that inhibits downstream translation in transfection assays and maymediate regulation of gp48 translation during infection, possibly bedelaying such translation until conditions for it are most favorable. Anessential element of the cis-acting sequence is an upstream open-readingframe in the 5′-UTR, that is, a short coding sequence beginning with anAUG that is not the initiator AUG for the gp48 protein. Further evidencesuggests that a cellular-factor may be activated during cytomegalovirusinfection and alleviate the inhibitory effects of the upstreamopen-reading frame. The latter may thus represent another viral nucleicacid sequence which at the correct stage of the viral replicative cycleis responsible for preferential translation of a viral RNA.

[0230] IRES elements and the influenza virus 5′-UTR are discussed indetail herein but are only examples of a broader class of viral nucleicacid sequences responsible for preferential translation of viral RNAover host RNA. The present invention applies equally well to other viralnucleic acid sequences within this broad class. A variety of proceduresare available to those skilled in the art which enables them to identifyother such viral nucleic acid sequences and to design methods forselecting agents which can prevent these sequences from mediatingpreferential translation of viral RNAs. In general, the steps involvedinclude: to ascertain whether viral RNAs are being preferentiallytranslated during infection by a given virus; to determine whetherspecific viral nucleic acid sequence(s) mediate the preferentialtranslation; to identify other cellular and/or viral componentsinvolved; to characterize the interaction between the viral nucleic acidsequence(s) and these components; and to design a screening method inwhich disruption or moderation of the effect of the viral nucleic acidsequence(s) can be detected. Not all of these steps may be required, andthe steps may be performed in any order depending on the nature of theresults obtained. The specific details of these steps now follow. Manyof the procedures used are collected in such reference texts as AusubelF et al. (eds) Current Protocols in Molecular Biology,Wiley-Interscience, New York, 1991, and Sambrook et al., MolecularCloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0231] Preferentially Translated Viral RNAs

[0232] Several methods can be used to determine whether viral RNAs arepreferentially translated during infection by a particular virus. Oneapproach is to incubate uninfected and infected cells in the presence ofa labeled amino acid, and to examine the labeled proteins synthesized inthe two different types of cell. The labeled amino acid may typically beone that includes a radioactive isotope, such as [³⁵S]methionine,[³⁵S]cysteine, [³H]leucine, or [¹⁴C]leucine. As an alternative toincubating intact cells with radiolabeled substrates, extracts can bemade from uninfected and infected cells and utilized in in vitrotranslations with these substrates, examining the translation ofendogenous mRNAs or test mRNAs added to the cell extracts. A test viralRNA and a test cellular mRNA can for example be added to extracts madefrom either uninfected or infected cells and the translation of eachtype of RNA in each type of extract be studied.

[0233] Whether the experiments are performed in cells or in cellextracts, uptake of the labeled precursor into protein may be followedby measuring the incorporation of label intotrichloroacetic-acid-precipitable protein. The types and relativequantities of proteins synthesized can also be assessed by usingpolyacrylamide gel electrophoresis to separate these proteins. Theseparated proteins can be detected by autoradiography or byfluorography, for example with a Phosphor Imager™ device, and analyzedby comparison with standard labeled proteins of known molecular weightsincluded on the same polyacrylamide gel during electrophoresis. Viralproteins can be recognized in this analysis from a knowledge of theirmolecular weights. If these are not known, it may be possible to inferwhich of the proteins observed are viral proteins from the pattern ofbands on the gels from uninfected and infected cells (or cell extracts):bands which are absent in the pattern from uninfected cells butsignificant in the pattern from infected cells are likely to representviral proteins. Indeed, significant changes in band pattern are usuallystrongly indicative of the preferential translation of viral RNAs.

[0234] Viral proteins in electrophoresis gels can also be identified byother means, for example by Western blotting. This involves transferringthe band pattern from the electrophoresis gel to a solid support andthen exposing the transferred pattern to an antibody or antibodiesspecific for a viral protein or proteins, detecting bound antibody withany of several antibody-detecting procedures known to those skilled inthe art. By performing a parallel Western blot using an antibody orantibodies specific for a known cellular protein or proteins, it ispossible to compare the synthesis of viral and cellular proteins inuninfected and infected cells. If viral proteins are synthesized insignificantly greater quantity than cellular proteins in infected cells,and/or if any or many or all cellular proteins are synthesized inreduced quantities in infected cells compared with uninfected cells,this indicates preferential translation of viral RNAs.

[0235] In an alternative approach using antibodies, antibodies specificfor viral and cellular proteins can be used to immunoprecipitate orotherwise separate their respective antigens prior to electrophoresis ora quantitative measurement such as measurement, of incorporatedradioactivity or enzyme activity or binding activity or agglutinationactivity can be used. Such determinations are informative to establishwhether preferential translation of viral RNAs is occurring in infectedcells.

[0236] The functions of viral and cellular proteins can also be assayedwithout prior immunoseparation of these proteins. The concentrations ofsuch proteins can also be determined by immunoassays or othercompetitive binding assays.

[0237] Another approach to identifying whether viral RNAs arepreferentially translated in infected cells is to perform what is knownas a transfection-infection assay. In an assay of this sort, a gene orcomplementary DNA (cDNA) which encodes a protein capable of beingassayed or detected is introduced into a cell by transfection, and thecell is also infected with the virus under study. In some assays thetransfected gene is a cellular gene or cDNA whose transcription willprovide an mRNA containing normal cellular translation sequences such as5′- and 3′-untranslated leaders and a poly(A) tail. In other assays thetransfected gene is a viral gene or cDNA whose transcription orreplication will provide an RNA containing normal viral translationsequences. If the protein encoded by the transfected viral gene isproduced in greater quantities in infected cells than the proteinencoded by the transfected,cellular gene, relative to the amounts ofthese proteins produced in uninfected cells, this indicates preferentialtranslation of viral RNAs in infected cells. It is also possible toperform these assays by transfecting both the cellular and the viralgenes into the same cell and infecting this cell with the virus understudy.

[0238] It will be evident to one skilled in the art thattransfection-infection assays can be replaced by similar assays in whichstable cell lines are used which express cellular or viral reporter geneconstructs. Such cell lines can be developed using selectable markergenes such as neo. With such a cell line the transfection step would beeliminated, and assays would simply involve infection of the stable cellline with the virus.

[0239] Examination of the RNAs present in uninfected and infected cellsmay also form a part of any investigation into whether viral RNAs arebeing preferentially translated. The presence and relativeconcentrations of viral and cellular RNAs can be studied by a variety ofprocedures known to those skilled in the art, such as Northern blothybridizations, nuclease protection assays, primer extension reactions,and the like.

[0240] Specific Viral Nucleic Acid Sequences Mediating PreferentialTranslation

[0241] Various approaches are available to determine whether specificviral nucleic acid sequences are responsible for the preferentialtranslation of viral RNAs. These include, but are not limited to,studies with chimeric RNAs having a detectable reporter polypeptidetranslationally linked to a viral nucleic acid sequence potentiallyresponsible for the preferential translation; studies of naturallyoccurring and laboratory mutants of viral nucleic acid sequences; andtransfection-infection assays.

[0242] A fruitful initial approach is often to construct chimeric RNAshaving the coding sequence for a detectable reporter polypeptide linkedto a viral nucleic acid sequence potentially responsible for thepreferential translation of viral RNAs. Production of the detectablereporter polypeptide is then examined in translation conditions underwhich this reporter will not be produced unless the viral nucleic acidsequences ensure its translation. As a control, production of thedetectable reporter polypeptide will also be examined under the sametranslation conditions from parallel constructs in which the reporter isnot linked to the viral nucleic acid sequences under test. As anadditional control, the chimeric RNA, or alternatively a second RNAadded to each test, may include the coding sequence for a seconddetectable reporter polypeptide distinguishable from the first andtranslationally linked to RNA sequences responsible for ensuring normaltranslation of cellular mRNAs.

[0243] In some cases the translation conditions used for the test willbe the translation conditions present in infected cells. In such casesthe test can be performed by introducing the chimeric RNA or a DNAsequence encoding it into cells which previously, concurrently orsubsequently are also infected with the virus under study. Thetransfection-infection assay described in more detail below is anexample of such a test. As an alternative to performing the test inintact cells, the translation conditions present in infected cells canbe reproduced in vitro by preparing extracts from infected cells andadding these to, or using them for, in vitro translations of thechimeric RNAs.

[0244] In other cases it may not be necessary to work with infectedcells or extracts made from them. In some cases the chimeric RNA can beconstructed in such as way that there will be limited or no productionof the detectable reporter polypeptide in uninfected cells or in vitrotranslation extracts from such cells unless the test sequence linked tothe coding sequence for the reporter allows preferential translation ofthe reporter. An example would be a chimeric RNA in which production ofthe detectable reporter polypeptide requires initiation of translationat an internal site within the RNA. In other cases it may be possible toadd an inhibitor to uninfected cells or extracts made from them whichblocks a step or pathway normally blocked during viral infection. Anexample would be the addition of cap analogs to inhibit cap-dependentinitiation of translation.

[0245] Detectable reporter polypeptides suitable for use in chimericRNAs or control RNAs include, but are not limited to, easily assayedenzymes such as β-galactosidase, luciferase, β-glucuronidase,chloramphenicol acetyl transferase, and secreted embryonic alkalinephosphatase; proteins for which immunoassays are readily available suchas hormones and cytokines; proteins which confer a selective growthadvantage on cells such as adenosine deaminase, aminoglycosidephosphotransferase (the product of the neo gene), dihydrofolatereductase, hygromycin-B-phosphotransferase, thymidine kinase (when usedwith HAT medium), xanthine-guanine phosphoribosyltransferase (XGPRT),and proteins which provide a biosynthetic capability missing from anauxotroph; proteins which confer a growth disadvantage on cells, forexample enzymes that convert non-toxic substrates to toxic products suchas thymidine kinase (when used with medium containing bromodeoxyuridine)and orotidine-5′-phosphate decarboxylase (when used with 5-fluorooroticacid); and proteins which are toxic such as ricin, cholera toxin ordiphtheria toxin.

[0246] Viral nucleic acid sequences responsible for preferentialtranslation of viral RNAs can also be identified by studies of naturallyoccurring and laboratory mutants. The latter may be constructed by avariety of procedures known to those skilled in the art, including butnot limited to chemical treatment with mutagens, and the use ofmolecular biology techniques to generate insertions, substitutions,deletions and point mutations in viral nucleic acid sequences. Theimpact of various mutations on the preferential translation of viralproteins can then be assessed by the methods described above forstudying such preferential translation.

[0247] In a related approach, the mutational analysis can be performedon viral nucleic acid sequences that are translationally linked tocoding sequences for detectable reporter polypeptides within chimericRNAs of the type described above. The impact of mutations within theviral nucleic acid sequences can be assessed by examining the productionof the detectable reporter polypeptide under translation conditionswhich require a functioning viral nucleic acid sequence for the reporterto be synthesized. This approach can be particularly productive fordetailed mapping and characterization of the regions within a viralnucleic acid sequence which are important for its function in ensuringpreferential translation of viral RNAs.

[0248] Transfection-infection assays are another tool which can be usedto identify viral nucleic acid sequences which ensure preferentialtranslation of viral RNAs. As explained above, such assays involve theintroduction into a cell by transfection of a gene or complementary DNA(cDNA) which encodes a reporter protein that can be assayed or detected,and infection of this cell with the virus under study. To use this typeof assay to identify a viral nucleic acid sequence conferringpreferential translation, different chimeric constructs would be madewith the same reporter gene/protein. In some constructs the RNAstranscribed from this gene will contain normal cellular translationsequences, and in others they would contain viral nucleic acid sequencesbelieved to be responsible for preferential translation of viral RNAs.If production of the reporter protein in infected cells is lower fromRNAs containing cellular translation sequences than it is from RNAscontaining viral nucleic acid sequences, this indicates that the viralsequences in question are capable of mediating preferential translation.

[0249] It will be evident to one skilled in the art that this type oftransfection-infection assay can also be used to analyze mutations madein viral nucleic acid sequences in order to map and characterize theprecise regions of these sequences responsible for mediatingpreferential translation.

[0250] 5′-untranslated leader sequences potentially containing sequenceelements useful in the practice of this invention are known for a numberof viruses and viral strains, as detailed in the following publications:

[0251] Coxsackievirus

[0252] Jenkins O., 1987, J. Gen. Virol 68, 1835-1848

[0253] Ilzuka et al., Virology 156, 64.

[0254] Hughes et al., 1989, J. Gen. Virol. 70, 2943-2952.

[0255] Chang et al.,, 1989, J. Gen. Virol. 70, 3269-3280.

[0256] Chang et al., 1989, J, Gen. Virol. 70, 3269-3280.

[0257] Lindberg et al.,1987 Virology 156, 50.

[0258] Tracy et al., 1985 Virus Res. 3, 263-270.

[0259] Hepatitis A Virus

[0260] Cohen J I et al.,1987, Proc. Natl. Acad. Sci. USA 84, 2497-2501.

[0261] Paul et al., 1987, Virus Res. 8, 153-171.

[0262] Cohen et al., 1987, J. Virol. 61, 50-59.

[0263] Linemeyer et al.,1985 J. Virol. 54, 252.

[0264] Najarian et al., 1985 Proc. Natl. Acad. Sci. USA 82, 2627

[0265] Baroudy B M et al., 1985 Proc. Natl. Acad. Sci. USA 82,2143-2147.

[0266] Poliovirus

[0267] Racaniello & Baltimore 1981 Proc. Natl. Acad. Sci. USA 78,4887-4891;

[0268] Stanway G et al.,1984 Proc. Natl. Acad. Sci. USA 81, 1539-1543.

[0269] La Monica N et al., 1986 J. Virology 57, 515.

[0270] Hughes P J et al., 1986 J. Gen. Virol. 67, 2093-2102.

[0271] Hughes P J et al., 1988 J. Gen. Virol. 69, 49-58.

[0272] Ryan M D et al.,1990 J. Gen. Virol 71, 2291-2299.

[0273] Pollard et al., 1989, J. Virol.,63, 4949-4951.

[0274] Nomoto et al., 1982 Proc. Natl. Acad. Sci. USA 79, 5793-5797.

[0275] Toyoda et al., 1984, J. Mol. Biol. 174, 561-585.

[0276] Rhinovirus

[0277] Deuchler et al., 1987 Proc. Natl. Acad. Sci. USA 84, 2605-2609.

[0278] G. Leckie, Ph.D.thesis University of Reading, UK.

[0279] Skern T et al., 1985, Nucleic Acids Res. 13, 2111.

[0280] Callahan P et al., 1985 Proc. Natl. Acad. Sci. USA 82, 732-736.

[0281] Stanway et al.,1984 Nucl. Acids Res. 12, 7859-7875.

[0282] Bovine enterovirus

[0283] Earle et al., 1988, J. Gen. Virol. 69, 253-263.

[0284] Foot-and mouth disease virus

[0285] Forss et al., 1984, Nucleic Acids Res. 12, 6587.

[0286] Beck et al., 1983, Nucleic Acids Res. 11, 7873-7885.

[0287] Villanueva et al., 1983, Gene 23, 185-194.

[0288] Beck et al., 1983, Nucleic Acids Res. 11, 7873-7885.

[0289] Carroll A R et al., 1984 Clarke Nucleic Acids Res. 12, 2461.

[0290] Boothroyd et al., 1982, Gene 17, 153-161.

[0291] Boothroyd et al., 1981 Nature, 290, 800-802.

[0292] Robertson et al., 1985, J. Virol. 54, 651.

[0293] Wendell et al., 1985 Proc. Natl. Acad. Sci. USA 82, 2618-2622.

[0294] Enterovirus Type 70

[0295] Ryan, M D et al. 1989 J. Gen. Virol.

[0296] Theiler's murine encephalomyelitis virus

[0297] Ohara et al., 1988, Virologyl64, 245.

[0298] Peaver et al., 1988, Virology 165, 1.

[0299] Peaver et al., 1987, J. Virol. 61, 1507.

[0300] Encephalomyocarditis Virus

[0301] Palmenberg et al., 1984 Nucl. Acids Res. 12, 2969-2985.

[0302] Bae et al., 1989 Virology 170, 282-287.

[0303] Hepatitis C Virus

[0304] Inchauspe et al., 1991 Proc. Natl. Acad. Sci. USA 88, 10293.

[0305] Okamoto et al., 1992, v 188, 331-341

[0306] Kato et al., 1990, Proc. Natl. Acad. Sci. USA 87, 9524-9528

[0307] Takamizawa et al., 1991, J. Virology 65, 1105-1113

[0308] Okamoto et al., 1991, J. Gen. Virol 72, 2697-2704

[0309] Choo et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2451-2455

[0310] Han et al., 1991 Proc. Natl. Acad. Sci. USA 88, 1711-1715

[0311] Influenza Virus

[0312] Fiers W et al., 1981, J. Supramol Struct Cell Biochem (Suppl 5),357.

[0313] The sequence of the 5′-UTR isAGCAAAAGCAGGGUAGAUAAUCACUCACUGAGUGACAUCAAAAUC. The 12 nucleotidesunderlined are conserved in all influenza mRNAs.

[0314] Also known is the sequence of hepatitis B virus: Galibert et al.,1979 Nature 281, 646-650.

[0315] Identification of Other Components

[0316] Once a viral nucleic acid sequence has been identified asresponsible for preferential translation of viral RNAs, a variety ofprocedures are available to identify cellular and/or viral componentsinvolved in the action of this viral nucleic acid sequence.

[0317] Proteins or other macromolecules which bind directly to thisviral nucleic acid sequence are clearly of particular interest. Onemethod to identify such proteins or macromolecules is to use gelretardation assays. In such assays, an RNA species consisting of orcontaining the viral nucleic acid sequence would be prepared in labeledform, for example by transcription in the presence of labelednucleotides from an appropriate DNA constructed for the purpose. Samplesof the labeled RNA would then be brought into contact with cellextracts, for example extracts made from infected and uninfected cells,and subjected to electrophoresis alongside samples of the labeled RNAwhich had not been placed in contact with such cell extracts. A decreasein mobility of the labeled RNA which had been in contact with cellextracts would indicate the presence in those extracts of proteins ormacromolecules which bind to the RNA.

[0318] Another method to identify such proteins is UV-cross-linking.This also utilizes labeled RNA consisting of or containing the viralnucleic acid sequence of interest. The labeled RNA is first incubatedwith cell extracts from uninfected or infected cells, and anyRNA-protein complexes which form are then cross-linked by exposure toultraviolet light, for example light of wavelength 254 nm. RNA notinvolved in cross-linked complexes is removed by nuclease treatment, andthe complexes are subjected to SDS-polyacrylamide gel electrophoresisfollowed by autoradiography or fluorography to determine the molecularweights of proteins/macromolecules involved in the complexes. Theproteins which become cross-linked to labeled RNA can also be examinedby immunochemical procedures such as Western blotting orimmunoprecipitation.

[0319] If an antibody is made or available against a protein suspectedof involvement in the action of a viral nucleic acid sequence whichmediates preferential translation, evidence for the involvement of thisprotein can also be gained by testing the effect of this antibody ontranslation mediated by the viral sequence.

[0320] Another approach to identifying cellular or viral proteins whichinteract with a specific viral nucleic acid sequence is to prepare thissequence in labeled form and use it as a probe to screen “expressionlibraries” of cellular or viral genes/cDNAs. Such libraries areconstructed in such a way that the protein encoded by each cloned geneor cDNA is expressed within the clone that contains it; they are oftenmade in the Lambda gt11 vector or similar vectors. In the present case,if a clone is producing a protein which interacts with the viral nucleicacid sequence then labeled probe should adhere specifically to thatclone.

[0321] Labeled viral nucleic acid sequences can also be used as probesto analyze proteins which have been separated by electrophoresis andtransferred to a membrane support such as a nitrocellulose membrane.Bands to which the labeled probe adheres represent proteins capable ofbinding the viral nucleic acid sequences.

[0322] In a further approach, a viral nucleic acid sequence known to beresponsible for mediating preferential translation can be used as anaffinity ligand to separate proteins which bind to it. Thus, the viralnucleic acid sequence can be attached to a chromatography support andused to separate proteins of interest from a cell extract by affinitychromatography. Alternatively, the viral nucleic acid sequence can belabeled with a capture group enabling it to be captured from solutionusing an appropriate capture reagent. Proteins which bind to the viralnucleic acid sequence can then be captured along with this sequence. Thecapture group used to label the viral nucleic acid sequence can, forexample, be biotin (in which case the capture reagent would be avidin orstreptavidin) or digoxigenin (in which case the capture reagent would bean antibody specific for this hapten) Labeling of the nucleic acid withthe capture group can be achieved by incorporation of label-bearingribonucleotides during transcription of the nucleic acid from anappropriate template, or if the capture group is biotin by labeling witha photoactivable reagent such as photobiotin.

[0323] Sucrose density gradients can also be used to identify individualproteins or complexes of proteins and/or other macromolecules involvedin the preferential translation of viral RNA mediated by a viral nucleicacid sequence. Thus, for example, a viral RNA can be incubated with aribosomal salt-wash or other fraction prepared from a cell extract, andcomplexes detected by sedimentation on a sucrose density gradient. Todetermine whether such a complex is involved in preferential viraltranslation, it can be formed in the presence of unlabeled viral RNA,collected from the sucrose gradient, dissociated from the RNA bytreatment with micrococcal nuclease, and further treated withethyleneglycol-bis-(B-aminoethyl ether) N,N′-tetra acetic acid (EGTA) toinhibit the micrococcal nuclease. The dissociated components can then beused to supplement an in vitro translation system to determine whetherthey improve or enhance the translation of viral RNA under appropriateconditions.

[0324] To determine whether proteins are required for the formation ofcomplexes so identified, the cell extract can be mixed with labeledviral RNA and the mixture treated with proteinase K immediately prior tosedimentation on the sucrose gradient. If protease-sensitive componentsare required for the integrity of the complex the labeled RNA in anuntreated sample will sediment more quickly than the RNA in a sampletreated with proteinase K before loading on the gradient.

[0325] To determine whether specific proteins such as known translationfactors are involved in the formation of a complex, antibodies specificfor known proteins can be added to the complex-formation mixture andtheir impact on complex formation examined by analysis on sucrosegradients. Other protein components of the particle can be identified byforming the complex with labeled viral RNA, sedimenting it on a sucrosegradient, collecting the radioactive fractions corresponding to thecomplex, cross-linking the proteins to the RNA using ultraviolet light,degrading the RNA, and separating any labeled proteins on aSDS/polyacrylamide gel. The complex may be treated with an iodinationreagent prior to cross-linking to provide cross-linking sites onproteins which otherwise would not become cross-linked. As analternative to cross-linking, it is usually possible because of the highcapacity of sucrose gradients to isolate a complex in sufficientquantity to recover its protein components by precipitation with acetoneor trichloroacetic acid, prior to analysis by polyacrylamide gelelectrophoresis.

[0326] It is also possible that a complex formed with viral RNA willcontain other nucleic acids in addition to the viral RNA used to formit. Such nucleic acid components in the complex can be detected byforming the complex with unlabeled viral RNA, collecting the fractionscorresponding to the complex from a sucrose gradient, extracting themwith phenol, precipitating nucleic acids with ethanol, end-labeling themat the 5′-end using [gamma-³²P]ATP or at the 3′-end using [5′-³²P]pCp or[a-³²P]ddATP, and identifying labeled nucleic acids by electrophoresison a 10% denaturing polyacrylamide gel (for shorter molecules) and a 1%agarose gel (for longer nucleic acids). Any end-labeled nucleic acidfound, other than the viral RNA, can then be sequenced by enzymatic orchemical methods.

[0327] Protein-protein interactions play an important role in theregulation of translation and in the preferential translation of viralRNAs. Proteins involved in important interactions with other proteinscan be identified using a yeast genetic system known as the two-hybridsystem (Fields & Song, 1989, Chien et al., 1991). This requires theavailability of a gene or cDNA encoding one of the two proteins whichinteract with each other. In the present case this gene or cDNA can beobtained by any of the several methods described in the preceding text.This gene or cDNA would be cloned into a specific plasmid in such a waythat it is expressed fused to the DNA-binding domain of a yeasttranscriptional activator such as GAL4 which has two separable andfunctionally essential domains, one for DNA-binding and the other fortranscriptional activation. In parallel, genes or cDNAs encodingputative binding partners of the known component are cloned in such away that each putative partner is expressed fused to the transcriptionalactivation domain of the same DNA-binding protein. Introduction of bothtypes of fusion into the same yeast cell results in generation offunctional DNA-binding protein only if the fusion partners of the twodomains of this protein interact with one another closely enough tobring together its two separately-expressed domains. Clones whichproduce such functional DNA-binding protein can be selected very easilyby plating them on a medium which requires the yeast to produce anenzyme that is under the control of the DNA-binding protein. The gene orcDNA for the partner which binds to the previously identified componentcan then be recovered from yeast clones which grow on the selectivemedium.

[0328] The power of yeast genetics can also be harnessed in a ratherdifferent approach to identifying components which interact with viralnucleic acid sequences of interest. In this approach, a construct wouldbe made initially in which a sequence encoding a reporter polypeptideeasily detectable in yeast would be coupled to the viral sequence ofinterest. This construct would be introduced into a suitable yeaststrain and conditions established under which the reporter polypeptideis synthesized. The yeast strain would then be subjected to mutagens,and mutants isolated in which the reporter polypeptide is no longersynthesized. Each such mutant would then be used as the host in theconstruction of a complete library of yeast genes, and the library wouldbe screened to identify clones which express the reporter polypeptidebecause the cloned gene they contain is complementing the mutation inthe mutant host strain. This cloned gene is then analyzed to determinewhether it encodes a product that interacts with the viral sequencecoupled to the coding sequence for the reporter polypeptide. This can beachieved, for example, by using the cloned gene to direct the synthesisof its product from a transcription or expression vector and thenexamining the interaction of the gene product with the viral nucleicacid sequence, for example by any of the methods described above. Thecloned gene can also be used as a means to identify homologous human orviral gene(s). It can, for example, be labeled and used as ahybridization probe to screen a human or viral gene/cDNA library, orsequenced in order to provide the sequences for amplification primerswhich can be used to amplify the corresponding gene or mRNA from humancells or viruses or viral-infected cells by the polymerase chainreaction. Isolation of human or viral gene(s) can also be accomplisheddirectly, by making a library of human or viral genes in the mutatedyeast strain which no longer produces the reporter polypeptide, andlooking for complementation of the mutation by a human or viral gene.

[0329] Reporter polypeptides suitable for use in this genetic approachinclude, but are not limited to, easily assayed enzymes such asβ-galactosidase, luciferase, and chloramphenicol acetyl transferase;proteins for which immunoassays are readily available such as hormonesand cytokines; proteins which confer a selective growth advantage oncells, and in particular proteins that provide a biosynthetic capabilitymissing from an auxotroph, such as the products of the LEU2, URA3, HIS3and TRP1 genes; and proteins which confer a growth disadvantage oncells, for example, enzymes that convert non-toxic substrates to toxicproducts, such as the URA3 gene product (orotidine-5′-phosphatedecarboxylase) when supplied with 5-fluoroorotic acid.

[0330] An alternative but related approach using reporter geneconstructs in yeast is to introduce defined mutations in the viralnucleic acid sequence which is translationally linked to the reporterpolypeptide, such that this reporter is no longer produced in a givenstrain of yeast. By plating these yeast on a selective medium requiringproduction of the reporter polypeptide for growth, spontaneous mutantscan be selected which are able to overcome the mutation within the viralnucleic acid sequence. Gene libraries can then be made from these mutantyeast using the original strain as host, and complementation used toselect the genes responsible for overcoming the defect in the viralnucleic acid sequence.

[0331] Another approach to identifying cellular or viral componentsinvolved in the preferential translation of viral RNAs is to fractionateextracts from uninfected and infected cells based on their ability toinhibit or stimulate the translation in vitro of a detectable reporterpolypeptide from a chimeric RNA containing the coding sequence for thisreporter polypeptide linked to viral nucleic acid sequences responsiblefor preferential translation. Thus, extracts from uninfected andinfected cells are initially added to parallel but separate in vitrotranslation reactions and their effects on these reactions compared. Thetwo types of extract are then fractionated in parallel using a varietyof procedures known to those skilled in the art, and correspondingfractions from the two extracts is tested in parallel for their effectson in vitro translation reactions. Fractions found to contain atranslation-affecting component from one type of cell (infected oruninfected) are then fractionated further in parallel with thecorresponding fractions from the other type of cell (uninfected orinfected), and the new fractions obtained from this next round offractionation are also tested in in vitro translation reactions.Repeated iterations of this fractionation and testing procedure willeventually provide a relatively purified fraction containing acomponent(s) involved in the preferential translation of viral RNAs.

[0332] A similar approach to fractionation can be adopted using gelretardation assays as described above rather than in vitro translationsto monitor the progress of the fractionation.

[0333] Fractionation methods which can be used in this approach include,but are not limited to, centrifugation, ammonium sulfate precipitation,other differential precipitations, gel filtration, ion exchangechromatography, hydrophobic interaction chromatography, reverse phasechromatography, affinity chromatography, differential extractions,isoelectric focusing, electrophoresis, isotachophoresis, and the like.

[0334] Cellular or viral components involved in preferential translationof viral RNAs and isolated by any of the aforementioned fractionationapproaches can be utilized to help clone or identify the gene(s) whichcode for these components. If, for example, the component isolated is aprotein, its amino acid sequence or a part of that sequence can bedetermined by well known protein sequencing methods, and the sequenceinformation obtained can be used to predict the sequence ofoligonucleotides (which can be used as reverse transcriptase primers forcDNA synthesis or as amplification primers for the polymerase chainreaction, or as hybridization probes for screening gene/cDNA libraries).Alternatively, the isolated component can be used as an immunogen toraise antibodies against the component, which antibodies can then beused to screen cDNA expression libraries to identify clones encoding thecomponent. Antibodies can also be raised by synthesizing a short peptidecorresponding to part or all of any amino acid sequence determined fromthe isolated component, and using this peptide as immunogen. Thepeptide-induced antibodies can be used to screen cDNA expressionlibraries, or to affinity-purify the component in larger quantitiesenabling more extensive sequence determination, and thus providing moreextensive information on which to base a cloning strategy.

[0335] From this description it should be evident that a wide variety ofmethods are available to someone skilled in the art to identify cellularor viral components which interact with a viral nucleic acid sequenceresponsible for preferential translation of viral RNAs.

[0336] Characterization of Interactions

[0337] Many different methods are available to characterize theinteractions between cellular and/or viral components and viral nucleicacid sequences responsible for preferential translation. The methodsdescribed above for detecting these interactions can, for example, beused to analyze their susceptibility to changes in pH, ionic strength,temperature, the nature and mixture of anions and cations present, therelative concentrations of the cellular and/or viral components and thetest nucleic acid, the absolute concentrations of these components andnucleic acid, the availability of cofactors, the availability of anenergy source, the presence or absence of lipids, of nucleic acids, ofcarbohydrates, of other proteins, and/or of any other additives.Similarly, these methods can be used to examine the susceptibility ofthe interaction to treatment of one or more of the interacting materialswith chemicals or enzymes that cause modifications. A protein found tointeract with a viral nucleic acid sequence can, for example, be treatedwith alkylating agents, oxidizing agents, reducing agents, or otheragents which cause chemical modifications, or with enzymes thatphosphorylate, dephosphorylate, glycosylate, deglycosylate, add lipidside-chains, remove lipid side-chains, or cause other enzymaticmodifications.

[0338] Also informative is the effects of truncations, additions,substitutions, deletions, inversions and point mutations in the viralnucleic acid sequence and/or cellular components and/or viral componentswhich interact with it. Such structural alterations can be generated bytreatment of the respective materials with cleavage enzymes such asproteases, endoribonuclease and endodeoxyribonucleases, with editingenzymes such as DNA polymerases, with joining enzymes such as RNAligases, DNA ligases, and RNA splicing enzymes, with copying enzymessuch as DNA polymerases, RNA polymerases, and reverse transcriptases,with end-specific degrading enzymes such as 5′-exonucleases,3′-exonucleases, aminopeptidases and carboxypeptidases, with enzymesthat can add extensions to ends such as terminal deoxynucleotidyltransferase and poly(A) polymerase, and so on. Alternatively,structurally altered viral nucleic acid sequences and/or viralcomponents and/or cellular components can be generated by makingappropriate alterations to cloned genes and expressing these genes inintact cells or in in vitro systems. Thus, the use of restrictionenzymes, ligases, linkers, adapters, reverse transcriptases, DNApolymerases, RNA polymerases, polymerase chain reactions, site-directedmutagenesis, and randomized mutagenesis make it possible to generate anenormous spectrum of structurally altered forms of biomolecules whichinteract with one another. These structural alterations can then betested in the array of methods previously described to determine whetherthe alterations change or abolish the interaction between differentsequences and components and/or the impact of these sequences andcomponents on translation.

[0339] The interaction between a cellular or viral protein and a viralnucleic acid sequence can also be studied using methods known asfootprinting. In such methods, the nucleic acid sequence and protein areallowed to interact with one another, and a reagent capable of cleavingthe nucleic acid, such as a nuclease, is then added. Regions of thenucleic acid which interact with the protein will be inaccessible to thecleavage reagent and thus protected from its action. In a typicalfootprinting procedure, the nucleic acid is labeled at one end with adetectable label, such as a phosphate group containing ³²P, and theoutcome of the procedure is assessed by denaturing the products of thecleavage reaction, subjecting them to electrophoresis in apolyacrylamide sequencing gel, and detecting them by autoradiography orfluorography. The results obtained are compared with those from acontrol experiment in which the labeled nucleic acid had not beensubjected to interaction with the protein. In the latter case, cleavagesites should be distributed relatively evenly throughout the nucleicacid and a “ladder” of bands will be observed, each rung on the ladderrepresenting cleavage at a particular nucleotide in the sequence of thenucleic acid. In the test sample, however, some of the potentialcleavage sites should have been inaccessible to the cleavage reagentbecause of the binding of the protein to the viral nucleic acid, andbands corresponding to the protected sites should be missing from orunder-represented in the ladder of bands. The sections of the ladderwith under-represented bands can then be compared with the knownsequence of the viral nucleic acid to determine which regions of thisnucleic acid were interacting with the protein.

[0340] The cleavage reagents used in such procedures may be nucleases,more particularly ribonucleases when the test nucleic acid is RNA, orchemical reagents such as methylating reagents which predisposenucleotides they modify to subsequent cleavage with a second reagent, orfree radicals generated by reagents such as Fe²⁻ ions or the reagentknown as MPE.

[0341] Interactions between viral nucleic acid sequences and cellular orviral proteins can also be studied by a procedure known as aninterference assay which has some similarities to footprinting andyields similar information. This procedure utilizes a reagent which canchemically modify the nucleic acid sequence of interest so as to attachnew groups, such as methyl groups, to individual nucleotides in thenucleic acid. The procedure relies upon the attachment of such a groupto a specific nucleotide having two effects on that nucleotide:disruption of its ability to participate in an interaction with the testprotein, and predisposition of the nucleotide to cleavage with a secondreagent. To perform the procedure, the test nucleic acid sequence isend-labeled at one end and treated with the chemical modificationreagent under conditions such that only one nucleotide will be modifiedwithin each nucleic acid molecule, but that the position of thismodification within the length of the nucleic acid molecule will berandom (subject to any specificity of the modification reagent forspecific types of nucleotide, such as purines in general). The modifiednucleic acid is then allowed to interact with the test protein, andprotein-bound nucleic acid is separated from free nucleic acid, forexample by taking advantage of the reduced mobility of protein-boundcompared with free nucleic acid on electrophoresis gels. The nucleicacid associated with the test protein is then released and treated withthe second reagent, which cleaves this nucleic acid at sites modifiedwith the first modification reagent. If the latter is a reagent whichmethylates purine bases, for example, such as dimethyl sulfate, thecleavage can be accomplished with piperidine. The cleaved nucleic acidis then electrophoresed on a polyacrylamide sequencing gel, and thebanding pattern compared with that obtained by cleavage of labeled andmodified nucleic acid which was not subjected to interaction with thetest protein. Bands corresponding to cleavage at nucleotides involved inthe interaction with the test protein will be missing orunder-represented in the pattern obtained from the test sample, becausethe modification carried by these nucleotides prevented or reduced theirability to form complexes with the test protein.

[0342] From this description it should be evident that a variety ofmethods is available to someone skilled in the art to characterize theinteraction between a cellular or viral protein or component and a viralnucleic acid sequence responsible for preferential translation of viralRNAs.

[0343] Design of Methods to Screen Agents

[0344] Methods to screen agents for their ability to disrupt or moderatepreferential translation of viral RNAs can be designed without detailedknowledge of the precise interaction between the viral and cellularmaterials involved, although such a knowledge can certainly be helpful.Many of the numerous methods described above to identify the presence ofviral nucleic acid sequences which mediate preferential translation ofviral RNAs, to identify cellular or other viral components involved, andto characterize the interactions between these components and the viralnucleic acid sequences, can be readily adapted to detect interferencewith the aforementioned interactions or with the effects of theseinteractions.

[0345] Thus, for example, agents can be screened for their ability toprevent or reduce the binding between a cellular and/or viral proteinand a viral nucleic acid sequence as detected by a gel retardationassay. More generally, binding interactions between two or more partnerscan be measured in a variety of ways. One approach is to label one ofthe partners with an easily detectable label, place it together with theother partner(s) in conditions under which they would normally interact,perform a separation step which separates bound labeled partner fromunbound labeled partner, and then measure the amount of bound labeledpartner. The effect of a test agent included in the binding reaction canbe determined by comparing the amount of labeled partner which binds inthe presence of this agent to the amount which binds in its absence.

[0346] The separation step in this type of procedure can be accomplishedin various ways. In one approach, the unlabelled partner, or one of theunlabeled partners to the interaction is immobilized on a solid phaseprior to the binding reaction, and unbound labeled partner is removedafter the binding reaction by washing the solid phase. Attachment of theunlabeled partner to the solid phase is accomplished in various waysknown to those skilled in the art, including but not limited to chemicalcross-linking, non-specific adhesion to a plastic surface, interactionwith an antibody attached to the solid phase, interaction between aligand attached to the unlabeled partner (such as biotin) and aligand-binding protein (such as avidin or streptavidin) attached to thesolid phase, and so on.

[0347] Alternatively, the separation step can be accomplished after thelabeled partner had been allowed to interact with unlabeled bindingpartner(s) in solution. One example of such an approach is the gelretardation assay described earlier. Thus, in this case the labeledpartner is an RNA species consisting of or containing the viral nucleicacid sequence of interest, and the unlabeled partner is a preparationcontaining cellular and/or viral protein(s) which bind(s) to thisnucleic acid. The two partners is allowed to interact in solution, andany complexes of labeled RNA bound to unlabeled protein is detected bytheir slower mobility relative to unbound labeled RNA in electrophoresisgels. The amount of these complexes formed can be determined byquantitating the label associated with them. Test agents are judged bytheir ability to reduce or increase the amount of complexes formed.

[0348] Many other configurations are possible for binding assays inwhich the interaction between labeled and unlabeled partners occurs insolution and is followed by a separation step. In some cases sizedifferences between the labeled partner and the unlabeled partner can beexploited. Thus, for example, the separation can be achieved by passingthe products of the binding reaction through an ultrafilter whose poresallow passage of unbound labeled partner, but not of the unlabeledpartner or of the labeled partner once bound to its unlabeled partner.Alternatively, the products of the binding reaction can be passedthrough a gel filtration matrix which separates unbound labeled partnerfrom the unlabeled partner and from the labeled partner once bound tothe unlabeled partner. This can be achieved very conveniently bychoosing a gel filtration matrix whose exclusion limit is greater thanthe molecular size of one partner and less than the molecular size ofthe other; the larger partner will pass through the gel filtrationset-up in the void volume, while the smaller partner is elutedsignificantly later.

[0349] In another type of approach, separation can be achieved using anyreagent capable of capturing the unlabeled partner from solution, suchas an antibody against the unlabeled partner, a ligand-binding proteinwhich can interact with a ligand previously attached to this partner,and so on.

[0350] For any of the binding assays just described, the viral nucleicacid sequences can be provided by isolation and if necessaryfragmentation and/or fractionation of natural viral nucleic acids, or bycopying of such nucleic acids or fragments in vitro. A preferred routefor the provision of viral RNAs is to transcribe these from cloned viralgenes/cDNAs or fragments thereof, including labeled nucleotides duringthe transcription if the viral RNA is to be the labeled partner in thebinding reaction. The cellular and/or viral components for these bindingassays can be provided by preparation of extracts from uninfected and/orinfected cells, by partial or complete purification of the componentsfrom such extracts, by expression of the components from cloned genes,with or without purification of these components from the cells or invitro translation reactions in which they were expressed, and so on.Labeling of these components can be accomplished by a variety ofmethods, including but not limited to incorporation of labeledsubstrates such as radiolabeled amino acids during synthesis in cells orin vitro translation reactions, or by treatment with labeling reagentssuch as N-hydroxy succinimidyl esters containing biotin or other haptensor detectable ligands. Detection of the labeled partner in such assayscan be accomplished by a variety of procedures known to those skilled inthe art, including but not limited to autoradiography, fluorography,attachment of reporter polypeptides to ligands on one of the bindingpartners by means of antibodies, avidin, streptavidin or otherligand-binding proteins, and so on.

[0351] Binding assays are only one example of the types of assays whichcan be developed to screen agents for their ability to interfere in theinteractions between cellular or viral proteins or components and viralnucleic acid sequences responsible for preferential translation of viralRNAs. In another and preferred type of assay, agents is tested todetermine their impact on the translation of a detectable reporterpolypeptide from an RNA in which the coding sequence for the reporter istranslationally linked to a viral nucleic acid sequence responsible forpreferential translation of viral RNAs. Such assays were described insome detail above. Production of the detectable reporter polypeptide isexamined under translation conditions in which such production isdependent upon the viral nucleic acid sequence. As a control, thechimeric RNA or a second RNA included in each test can include thecoding sequence for a second detectable reporter polypeptidedistinguishable from the first and translationally linked to RNAsequences responsible for ensuring normal translation of cellular mRNAs.Test agents is examined for their ability to interfere with theproduction of the reporter polypeptide linked to the viral nucleic acidsequence without affecting production of the reporter polypeptide linkedto cellular translation sequences.

[0352] In some cases the translation conditions used for the test can bethe translation conditions present in infected cells. In such cases thetests can be performed by introducing the chimeric RNA or a DNA sequenceencoding it into cells which previously, concurrently or subsequentlyare also infected with the virus under study. The transfection-infectionassay described in more detail below is an example of such a test. As analternative to performing the test in intact cells, the translationconditions found in infected cells can be reproduced in vitro bypreparing extracts from infected cells and adding these to or using themfor in vitro translations of the chimeric RNAs.

[0353] In other cases it is not necessary to work with infected cells orextracts made from them, as for example in cases where the chimeric RNAcan be constructed in such a way that production of the detectablereporter polypeptide is dependent on a viral nucleic acid sequence evenin uninfected cells or in vitro translation extracts from such cells.This is the case for a chimeric RNA in which production of thedetectable reporter polypeptide requires initiation of translation at aninternal site within the RNA. In other cases it may be possible to addan inhibitor to uninfected cells or extracts made from them which blocksa step or pathway normally blocked during viral infection. An example isthe addition of cap analogs to inhibit cap-dependent initiation oftranslation.

[0354] Whichever approach is used, the tests can be performed in intactcells containing the chimeric RNAs, for example as the result oftranscription of an appropriate DNA introduced into the cells, or by invitro translation of these chimeric RNAs.

[0355] Detectable reporter polypeptides suitable for use in chimericRNAs or control RNAs include, but are not limited to, easily assayedenzymes such as β-galactosidase, luciferase, β-glucuronidase,chloramphenicol acetyl transferase, and secreted embryonic alkalinephosphatase; proteins for which immunoassays are readily available suchas hormones and cytokines; proteins which confer a selective growthadvantage on cells such as adenosine deaminase, aminoglycosidephosphotransferase (the product of the neo gene), dihydrofolatereductase, hygromycin-B-phosphotransferase, thymidine kinase (when usedwith HAT medium), xanthine-guanine phosphoribosyltransferase (XGPRT),and proteins which provide a biosynthetic capability missing from anauxotroph; proteins which confer a growth disadvantage on cells, such asenzymes that convert non-toxic substrates to toxic products such asthymidine kinase (when used with medium containing bromodeoxyuridine)and orotidine-5′-phosphate decarboxylase (when used with 5-fluorooroticacid); and proteins which are toxic such as ricin, cholera toxin ordiphtheria toxin.

[0356] Transfection-infection assays can also be used to identify agentswhich interfere in the interactions between cellular or viral proteinsor components and viral nucleic acid sequences responsible forpreferential translation of viral RNAs. As described above, such assaysinvolve the introduction into a cell by transfection of a gene orcomplementary DNA (cDNA) which encodes a detectable reporter polypeptidetranslationally linked to either a viral or a cellular translationsequence, and infection of this cell with the virus under study.Polypeptides linked to viral translation sequences are produced ingreater quantities in infected cells than polypeptides linked tocellular translation sequences. Test agents can be screened for theirability to reduce or abolish this disparity without affecting theproduction of the reporter polypeptide linked to the cellulartranslation sequences.

[0357] It will be evident to one skilled in the art thattransfection-infection assays can be replaced by similar assays in whichstable cell lines are used which express appropriate reporter geneconstructs. Such cell lines can be developed using selectable markergenes such as neo. With such a cell line the transfection step iseliminated, and assays would simply involve infection of the stable cellline with the virus.

[0358] In some cases the translation advantage conferred by a viralnucleic acid sequence may be so significant that it is observed evenwithout viral infection, when that sequence is introduced artificiallyinto a cell without other viral sequences. This is evidenced by superiortranslation in uninfected cells of a reporter polypeptide linked to theviral nucleic acid sequence as compared to the translation of the samepolypeptide linked to a cellular translation sequence. In such cases,test agents may be screened in uninfected cells by determining theirability to reduce the enhanced translation of the reporter polypeptidelinked to the viral sequence.

[0359] The above descriptions are provided by way of example and in noway limit the scope of the invention. It should be apparent that oneskilled in the art is able to choose from a wide. variety of methods toidentify viral nucleic acid sequences responsible for preferentialtranslation of viral RNAs, to identify other cellular and viralcomponents involved, to characterize the interactions between thevarious partners which enable preferential translation of viral RNAs,and to develop tests which can be used to screen agents for theirability to disrupt or abolish such interactions.

[0360] The following are examples of methods used to screen for agentsthat block activity of translational control elements.

[0361] Screening IRES Elements

[0362] Developing assays to screen for agents that block IRES elementactivity preferably requires constructing a dicistronic mRNAcharacterized by the presence of two different reporter genes, whereinthe translation of one gene is under IRES element control andtranslation of the other gene is under the control of the host-cell capstructure (m⁷GpppG) and cellular 5′-UTR sequence. Such a construct makesit possible to identify agents, using either cell-free or cell-basedassays, that block IRES element activity without adversely affecting theprocess that cells use to initiate translation of their own mRNA. Thus,the preferred embodiment of this invention enables the user to identifyagents that have the desired mechanism of action while simultaneouslyeliminating nonspecific and possibly toxic agents.

[0363] The reporter genes can be any genes that encode products that canbe conveniently and reliably detected. Commonly used detection methodsinclude, but are not limited to, incorporation of radioisotopes,chemiluminescence, bioluminescence, calorimetric techniques andimmunological procedures. Examples of appropriate reporter genes includeluciferase, chloramphenicol acetyl transferase, secreted embryonicalkaline phosphatase, β-galactosidase, and dihyrodofolate reductase.This list is merely illustrative and in no way limits the scope of theinvention since other suitable reporter genes will be known by thoseordinarily skilled in the art. The method(s) for detecting the reportergene products in the assay are preferably applied directly to thereactions or cells used to screen potential drug activity but, in alesser embodiment, can also be used in conjunction with techniques forfirst fractionating the reaction mixtures. Said techniques, used eithersingly or in combination, may include chromatography, electrophoresis,filtration, ultrafiltration, centrifugation, precipitation, extraction,complex formation or digestion.

[0364] The dicistronic reporter gene construct can be used for either invitro or in vivo agent screens. In the in vitro (cell-free) assayformat, the dicistronic mRNA construct is encoded by a plasmid DNAmolecule which directs transcription of the construct under the controlof a strong promoter, exemplified by the bacteriophage T7 or SP6promoters. When purified and transcribed in vitro with the homologousRNA polymerase (e.g. T7 or SP6) in the presence of pre-formed capstructures, the plasmid directs the synthesis of large amounts of“capped” dicistronic reporter construct that can be purified usingcommonly practiced techniques. This dicistronic mRNA is then used as atemplate in a eukaryotic in vitro translation system either purchasedfrom a commercial supplier or prepared according to procedures availablein the scientific literature.

[0365] Agents may also be tested in whole cells that contain the abovedicistronic reporter construct. Said construct is modified for use incultured eukaryotic cells by: 1) placing the transcription of theconstruct under the control of a strong eukaryotic viral promoter, suchas SV40, CMV or other promoters commonly used by those skilled in theart; 2) including splice signals such as SV40 splice signals to ensurecorrect processing and transport of RNAs made in the nucleus; and 3)including a polyadenylation signal such as the SV40 signal at the 3′ endof the construct so that the reporter mRNA will be synthesized as a 3′polyadenylated molecule.

[0366] A plasmid encoding the dicistronic construct can be used toestablish a transient expression assay for screening agents that blockIRES activity or, in the preferred embodiment, to establish a stablecell line for screening agents. The latter may be accomplished byincorporating into the plasmid harboring the dicistronic reporter geneconstruct any of several commonly used selectable markers, such as neo,in order to select and maintain those cells containing the assayplasmid. Alternatively, a stable cell line can be generated byco-transfecting the desired host cells with two plasmids, one containingthe selectable marker and the other containing the dicistronic reportergene construct. Selecting for cells in a co-transfection procedure thathave acquired one plasmid with a selectable marker is a commonly usedway known to those skilled in the art to purify cells which have takenup a second plasmid which lacks the benefit of a selectable marker.

[0367] Screening Viral 5′-UTRs

[0368] The assays to screen for agents that block 5′-UTR elementactivity preferably require constructing a test plasmid that directs thesynthesis of 2 mRNAs each representing a different reporter gene. Morespecifically, the synthesis of one reporter gene is under the control ofthe capped viral 5′-UTR and the synthesis of the second reporter gene isunder the control of the capped cellular 5′-UTR sequence. Such aconstruct makes it possible to identify agents, using either cell-freeor cell-based assays, that block viral 5′-UTR element activity withoutadversely affecting the process that cells use to initiate translationof their own mRNA. Thus, the preferred embodiment of this inventionenables the user to identify agents that have the desired mechanism ofaction while simultaneously eliminating nonspecific and possibly toxicagents from consideration.

[0369] The reporter genes can be any genes that encode products that canbe conveniently and reliably detected. Commonly used detection methodsinclude, but are not limited to, incorporation of radioisotopes,chemiluminescence, bioluminescence, calorimetric techniques andimmunological procedures. Examples of appropriate reporter genes includeluciferase, chloramphenicol acetyl transferase, secreted embryonicalkaline phosphatase, β-galactosidase, and dihyrodofolate reductase.This list is merely illustrative and in no way limits the scope of theinvention since other suitable reporter genes will be known by thoseordinarily skilled in the art. The method(s) for detecting the reportergene products in the assay are preferably applied directly to thereactions or cells used to screen potential drug activity but, in alesser embodiment, can also be used in conjunction with techniques forfirst fractionating the reaction mixtures. Said techniques, used eithersingly or in combination, may include chromatography, electrophoresis,filtration, ultrafiltration, centrifugation, precipitation, extraction,complex formation or digestion.

[0370] The reporter gene construct can be used for either in vitro or invivo agent screens. In the in vitro (cell-free) assay format, a chimericplasmid encodes each reporter gene under the control of a strongpromoter, exemplified by the bacteriophage T7 or SP6 promoters. Whenpurified and transcribed in vitro with the homologous RNA polymerase(e.g. T7 or SP6) and in the presence of pre-formed cap structures, theplasmid directs the synthesis of large amounts of “capped” reportermRNAs that can be purified using commonly practiced techniques. Thecapped mRNAs encoding each reporter gene are then used as templates in aeukaryotic in vitro translation system either purchased from acommercial supplier or prepared according to procedures available in thescientific literature.

[0371] Agents may also be tested in whole cells that contain the aboveconstruct carrying two reporter genes. Said construct is modified foruse in cultured eukaryotic cells by: 1) placing the transcription of thereporters under the control of strong eukaryotic viral promoters, suchas SV40, CMV or other promoters commonly used by those skilled in theart; 2) including splice signals, such as SV40 splice signals, for eachreporter to ensure correct processing and transport of RNAs made in thenucleus; and 3) including a polyadenylation signal, such as the SV40signal, at the 3′ end of each reporter gene so that the reporter mRNAwill be synthesized as a 3′ polyadenylated molecule.

[0372] The plasmid can be used to establish a transient expression assayfor screening agents that block viral 5′-UTR activity or, in thepreferred embodiment, to establish a stable cell line for screeningagents. The latter may be accomplished by incorporating into the plasmidharboring the two reporter genes any of several commonly used selectablemarkers, such as neo, in order to select and maintain those cellscontaining the assay plasmid. Alternatively, a stable cell line can begenerated by co-transfecting the desired host cells with two plasmids,one containing the selectable marker and the other containing the tworeporter genes. Selecting for cells in a co-transfection procedure thathave acquired one plasmid with a selectable marker is a commonly usedway known to those skilled in the art to purify cells which have takenup a second plasmid which lacks the benefit of a selectable marker.

[0373] Thus, as discussed above, some viruses contain 5′ untranslatedregions which include sequences providing a selective translationaladvantage to the associated RNA. These regions can be readily identifiedas exemplified herein, and used in assays for detection of specificantiviral agents. The following is an example of detection of such a5′-UTR in ‘flu virus, and is not limiting in this invention.

[0374] Influenza Virus

[0375] The ‘flu virus ensures selective translation of its own mRNAs bycausing host protein synthesis to undergo a rapid and dramatic shutoffsoon after ‘flu virus infection, but with ‘flu mRNAs still beingtranslated. one mechanism used to achieve this end (at least in the caseof a truncated ‘flu nucleocapsid protein (NP-S)) is a specific sequencein the 5′-untranslated region (UTR) of the mRNA. Translationalinitiation for the ‘flu mRNAs is still cap-dependent.

[0376] This sequence was identified using an assay termed atransection-infection assay. In this assay cells are transfected withcDNAs (genes) encoding a cellular protein which can be easily assayed,e.g., SEAP (secreted embryonic alkaline phosphatase), and then infectedwith ‘flu virus. If the SEAP has a normal cellular 5′-UTR, thesubsequent infection with ‘flu virus leads to a significant reduction inthe production of SEAP. If, however, the cellular 5′-UTR is replacedwith the 5′-UTR from the ‘flu mRNA encoding the NP-S protein, SEAPproduction continues unabated after ‘flu infection. This demonstratesthat the ‘flu 5-UTR contains some sequence ensuring translation of mRNAwhich contains it. By placing progressively smaller pieces of the ‘flu5′-UTR upstream of the SEAP gene, it is evident that as few as 12nucleotides are required to mediate the protective effect.

EXAMPLE 13 Plasmid Construction

[0377] Plasmid pNP-UTR/SEAP contains the region encoding the 5′-UTR forthe nucleocapsid protein of influenza virus strain A/PR/8/34 linked tothe coding sequence and 3′-untranslated region for secreted embryonicalkaline phosphatase (SEAP). The 5′-UTR of the modified influenza NP-Sgene (Garfinkel and Katze, 1992) was amplified by the polymerase chainreaction, using primers that placed a Hind III site at one end of theamplified product and a Sph I site at the other. The amplified productwas electrophoresed on an agarose or polyacrylamide gel, stained withethidium bromide, visualized by ultraviolet light, then excised andpurified from the gel fragment. The purified product was ligated intoplasmid pBC12/CMV/SEAP (Berger et al., 1988, Gene 66, 1) which hadpreviously been digested with Hind III and Sph I. The resulting plasmidswere introduced into E. coli and clones selected which contained thedesired construct. Plasmid pSEAP-UTR/NP-S contains the region encodingthe 5′-UTR for the SEAP protein linked to the coding sequence and3′-untranslated region for the influenza NP-S protein. The latter is aderivative of the nucleocapsid (NP) protein of influenza virus strainA/PR/8/34 obtained by deleting 255 nucleotides from within the NP gene(Garfinkel and Katze, 1992). The deletion creates a modified NP-Sprotein which can be distinguished from the native NP protein ininfluenza virus-infected cells. pSEAP-UTR/NP-S was prepared using thesame basic outline described above for the pNP-UTR/SEAP constrict,amplifying the SEAP-UTR by the polymerase chain reaction and placing itupstream of the NP-S coding sequence using appropriate restrictionsites. Plasmid pSEAP-UTR/SEAP is the pBC12/CMV/SEAP of Berger et al.,1988.

EXAMPLE 14 Cells and Transfections

[0378] COS-1 cells are grown in monolayers in Dulbecco's modifiedEagle's medium (DMEM) containing 10% fetal calf serum, and transfectedusing the DEAE-dextran/chloroquine method (Cullen, 1987, MethodsEnzymol. 152, 684). Monolayers are washed once with prewarmed,serum-free DMEM. DNA is added in the same medium containing 250 μg/mlDEAE-dextran. After 2 hours incubation of the cells, chloroquine isadded to a final concentration of 80 μM, and the cells incubated for afurther 2 hours. The transfection mixture is then removed and replacedwith a solution of 20% glycerol in HEPES-buffered saline. After 2minutes at room temperature, the cells are washes twice with Hanks'balanced salt solution (HBSS), then incubated in DMEM containing 10%fetal calf serum at 37° C.

EXAMPLE 15 Virus Infection

[0379] The WSN strain of influenza A virus are grown in Madin-Darbybovine kidney cells and titrated by plaque assay in Madin-Darby caninekidney cells (Etkind & Krug, 1975). Monolayers of COS-1 cells areinfected with influenza virus at a multiplicity of infection ofapproximately 50 plaque-forming units per cell. Cells transfected asdescribed in Example 2 are infected 40 hours after transfection. In allcases control cells are mock-infected with virus buffer at the same timethat test cells are infected with virus.

EXAMPLE 16 SEAP Assays

[0380] The activity of SEAP is assayed from the culture medium of cellscontaining the SEAP gene. Each assay is performed to measure the amountof SEAP secreted into the medium in a 30-minute period following achange of medium. Thus, to measure SEAP production at times 1 hour, 2hours, 3 hours, 4 hours, and five hours after infection, at each timepoint the medium in which the cells were infected is removed byaspiration, the cells are washed two to three times gently with warmmedium, and fresh serum-free medium (DMEM) is added. Incubation of thecells is continued for between 10 and 60 minutes, at which point 250 μlof medium is collected into a microcentrifuge tube and assayed for SEAPas described (Berger et al., 1988). The sample is heated to 65° C. for 5minutes and then centrifuged in a microfuge at top speed for 2 minutes.100 μl is removed to a fresh microcentrifuge tube, an equal volume of2×SEAP assay buffer is added (this buffer is 20 mM L-homoarginine, 1 mMMgCl₂, 2 M diethanolamine pH 9.8; 1 ml is prepared fresh each time bymixing 200 μl of 100 mM L-homoarginine, 5 μl of 0.2 M MgCl₂, 500 μl of 2M diethanolamine pH 9.8 which is stored in the dark, and 295 μl of H₂O),the sample is vortexed, and transferred to one well of a 96-wellmicrotiter plate. The plate is incubated at 37° C. for 10 minutes, afterwhich 20 μl of substrate solution (120 mM p-nitrophenylphosphate (Sigma,cat. no. N-2765) made up in 1×SEAP assay buffer) is added to each welland mixed with its contents by pipetting up and down. A readings aretaken at times zero, 30″, 1′, and at 1′ intervals thereafter. The plateis held at 37° C. between readings. The maximum linear rate of thereaction is determined by plotting A against time post-substrateaddition. Depending on the experiment, the maximum rate may be reachedanywhere between 2 minutes and 20 minutes after substrate addition.

EXAMPLE 17 NP-S Assays

[0381] To determine whether NP-S is being synthesized at a particulartime after infection, cells are labeled by incubation for 30 minuteswith [³⁵S]methionine (1200 μCi/ml) in methionine-free DMEM. Afterlabeling, cells are washed with ice-cold HBSS and lyzed in lysis buffer(10 mM Tris.HCl, pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1 mM dithiothreitol, 0.2mM phenylmethylsulfonyl fluoride, 100 units/ml aprotinin, 1% TritonX-100). The clarified lysate is diluted in buffer A (20 mM Tris.HCl, pH7.5, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mMphenylmethylsulfonyl fluoride, 100 units/ml aprotinin, 20% glycerol, 1%Triton X-100) and reacted twice, for 30 minutes each time, with proteinG-agarose that has been preloaded with monoclonal antibody 4F5 (from J.Yewdell, National Institutes of Health, although equivalent antibodiesare readily prepared). This antibody reacts with native NP protein butnot NP-S. The lysate is then reacted with protein A-agarose that has beepreloaded with pooled monoclonal antibody against NP (from R. Webster,St. Jude Children's Research Hospital, although equivalent antibodiesare readily prepared), which recognizes both NP and NP-S proteins.Precipitated products are washed four times in buffer A then three timesin buffer B (10 mM Tris.HCl, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 100units/ml aprotinin, 20% glycerol), boiled in an equal volume ofelectrophoresis buffer, and separated by electrophoresis on a gelcontaining 8% polyacrylamide, 0.3% bis and 4M urea. Radiolabeledproteins are then detected by autoradiography and quantitated by laserdensitometry.

EXAMPLE 18 Screening of Test Compounds

[0382] A concentrated solution of each test compound is prepared andvarious dilutions are made to produce test solutions at a range ofdifferent concentrations. Each test solution is then tested in theseries of experiments tabulated below by introducing it into the culturemedium at the time of infection, or at the equivalent time in controlsfor which no infection is performed: Plasmid used to Experimenttransfect cells Infection Agent 1 None No No 2 None No Yes 3pNP-UTR/SEAP No No 4 pNP-UTR/SEAP No Yes 5 pNP-UTR/SEAP Yes No 6pNP-UTR/SEAP Yes Yes 7 pSEAP-UTR/Np-s No No 8 pSEAP-UTR/NP-s No Yes 9pSEAP-UTR/NP-s Yes No 10 PSEAP-UTR/NP-s Yes Yes 11 pSEAP-UTR/SEAP No No12 pSEAP-UTR/SEAP No Yes 13 pSEAP-UTR/SEAP Yes No 14 pSEAP-UTR/SEAP YesYes

[0383] Samples are taken at various time points after infection andassayed for SEAP and NP-S as described in examples 16 and 17.

[0384] Screening Viral uORFs

[0385] This invention also encompasses methods for identifying agentsthat block viral uORF activity. These methods are essentially identicalto the viral uORF test systems except that they use leader sequencescontaining the target viral uORFs in place of the viral 5′-UTRsequences.

[0386] Libraries for Screening

[0387] The assays encompassed by this invention can be used to screenagent libraries to discover novel antiviral drugs. Such libraries maycomprise either collections of pure agents or collections of agentmixtures. Examples of pure agents include, but are not necessarilylimited to, proteins, polypeptides, peptides, nucleic acids,oligonucleotides, carbohydrates, lipids, synthetic or semi-syntheticchemicals, and purified natural products. Examples of agent mixturesinclude, but are not limited to, extracts of prokaryotic or eukaryoticcells and tissues, as well as fermentation broths and cell or tissueculture supernates. In the case of agent mixtures, the assays are notonly used to identify those crude mixtures that possess the desiredantiviral activity, but also the assays provide the means to purify theantiviral principle from the mixture for characterization anddevelopment as a therapeutic drug. In particular, the mixture soidentified can be sequentially fractionated by methods commonly known tothose skilled in the art which may include, but are not limited to,precipitation, centrifugation, filtration, ultrafiltration, selectivedigestion, extraction, chromatography, electrophoresis or complexformation Each resulting subfraction can be assayed for antiviralactivity using the original assay until a pure, biologically activeagent is obtained.

[0388] In preferred embodiments, the assays designed for detectingantiviral activity are used for automated, high-throughput drugdiscovery screens in conjunction with the above mentioned libraries. Theassays are performed in any format that allows rapid preparation andprocessing of multiple reactions such as in, for example, multi-wellplates of the 96-well variety. Stock solutions of the test agents aswell as assay components are prepared manually and all subsequentpipetting, diluting, mixing, washing, incubating, sample readout anddata collecting is done using commercially available robotic pipettingequipment, automated work stations, and analytical instruments fordetecting the signal generated by the assay. Examples of such detectorsinclude, but are not limited to, spectrophotomers, calorimeters,luminometers, fluorometers, and devices that measure the decay ofradioisotopes.

[0389] In another embodiment, the assays may be used to screen vastlibraries of random peptides or oligonucleotides produced by any of thetechniques already in the public domain or otherwise known to thoseskilled in the art. Because of their large size, these libraries arelikely sources of lead agents since they can contain from 10⁷-10¹⁰chemical entities. Screening libraries of this size requires allowingtest agents to bind to a molecular target in vitro, trapping theresulting complex in order to identify the specific lead agents thathave been bound, and then producing the lead) agents in greaterquantities for further development.

[0390] In the present invention, the molecular targets of choicecomprise those segments of viral RNA that insure preferentialtranslation of viral mRNA in virus-infected cells, as well as any viralor cellular protein(s) required by the viral RNA segment for thisfunction. Either the assay target or the library agents are immobilizedon a solid support so that the complexes formed between the moleculartarget and putative lead agents can be trapped and convenientlyseparated from unbound molecules. Amplification of the lead agents canbe done chemically (peptide or oligonucleotide synthesis, respectively,once the sequence of the test agent has been deduced), enzymatically(PCR amplification reactions in the case of oligonucleotides) orbiologically (propagation in E. coli of bacteriophage display vectors inthe case of peptides). The lead peptide or oligonucleotide agents may beultimately developed as drugs in and of themselves, or used forstructural modeling studies to develop small molecule mimics whichbecome the final drug.

[0391] The following broadly summarizes the main screening methodsuseful in this portion of the invention:

[0392] I. In vitro Binding Assays

[0393] Binding assays, described below, are biochemical methods thatmeasure the extent of interaction between any desired IRES element andviral and cellular proteins which bind to the IRES element to mediatetranslation under its influence. These techniques provide a basis forscreening libraries of synthetic, semi-synthetic, natural products orany mixtures thereof to identify potential anti-viral compounds. Suchcompounds, which interact with the IRES element and/or the IRES-bindingprotein(s), will block the formation of the requisite complex betweenthe IRES element and the viral or cellular protein(s) and thus willreduce or abolish IRES element activity. Compounds having suchproperties can be identified using a variety of in vitro binding assays.In these assays, is incubated with the IRES-binding protein and testcompound under conditions previously established to allow a stablecomplex to form between the IRES element and the binding protein in theabsence of the test compound. As described below, various methods areused to detect the extent of complex formation in the presence andabsence of the test compound.

[0394] A. In vitro IRES Element Binding

[0395] In this configuration, the selected IRES-binding protein, eitherviral or cellular, is first immobilized on a solid support using any ofthe techniques commonly used by those skilled in the art. Thesetechniques include, but are not limited to, contacting the purifiedbinding protein with a filter material made of nitrocellulose or a smallreaction vessel made of polystyrene whereupon the protein will beretained on these surfaces. In another embodiment, cell extracts ormixtures of proteins containing impure binding protein may be contactedwith a solid support to which is previously bound an antibody specificfor the IRES element-binding protein. The antibody traps the bindingprotein on the solid support such that washing the surface of thesupport with a buffered wash solution removes all unwanted proteins fromthe starting sample. Thus in one step the impure binding protein is notonly purified for assay purposes, but also is immobilized and ready foruse in the assay.

[0396] In order to measure the amount of complex formed between the IRESelement and the binding protein, IRES element preparations are usedwherein the element has been labeled in such a fashion to allowconvenient and sensitive detection of the element. Routine labelingprocedures may include chemical, enzymatic synthesis or biosynthesis ofthe IRES element in the presence of labeled precursors leading to theincorporation of the isotope throughout the IRES element. Also methodsfor end-labeling RNA molecules at their 5′ or 3′ ends with [³²P] arewell-known to those skilled in the art as are methods for derivatizingthe molecule with other readily detectable tags such as biotin. Whereasradioisotopically labeled IRES elements are detected by standard methodsincluding liquid scintillation spectrometry and radiographic imaging,immobilized IRES labeled with, for example biotin, can be detectedcalorimetrically or luminometrically by reacting the biotinylatedmolecule with a biotin-binding protein, such as streptavidin, and asecond biotinylated reporter molecule, such as alkaline phosphatase orluciferase, and incubating the resulting tripartite complex in thepresence of a substrate that is cleaved by the reporter molecule to forma colored or luminescent substance that can be detectedspectrophotometrically or with a luminometer.

[0397] In one form of the binding assay, the materials and techniquesdescribed above are employed to directly measure complex formationbetween the labeled IRES element and the binding protein in the presenceand absence of test compounds. More specifically, in one variation, thelabeled IRES element, binding protein, and test compound are incubatedin solution and the mixture is then passed through a nitrocellulosefilter which retains the binding protein because of the affinity of thefilter material for proteinaceous substances. Any labeled IRES elementbound to the binding protein will likewise be retained by the filter aspart of a binding protein-IRES element complex, whereas all unbound IRESelement will pass though the filter. Aliquots of buffered wash solutionmay be drawn through the filter several times to thoroughly wash thefilter free of unbound labeled IRES elements. Measurement of the amountof bound IRES element is achieved using any of the above detectionmethods. In a second variation, the binding protein is first immobilizedon the surface of a solid support which is typically configured asmultiple small reaction vessels (e.g., 96-well microtiter plate). Abuffered solution containing the labeled IRES element and test compoundis then added to the reaction vessel, incubated, and then the liquidcontents of the vessel are removed, and the vessel is finally rinsedseveral times with a wash buffer to remove the last traces of unboundIRES element. Measurement of the amount of bound IRES element in thepresence and absence of the test compounds is achieved using any of theabove detection methods.

[0398] The third form of the assay differs from the first two in thatthe IRES element RNA is itself immobilized on a solid support, which maycomprise a 96-well microtiter plate, and in this form is incubated withthe labeled binding protein and test compound under conditions whichallow formation of a stable complex between the IRES element and thebinding protein in the absence of the test compound. The IRES RNA may bebound directly to the solid support using methods commonly known tothose skilled in the art, or it may be attached to the surface with theaid of a polymeric linker which may support the IRES molecule at adistance from the surface of the support and in so doing may make theIRES RNA more accessible to binding by its recognition protein.Polymeric linkers may also comprise complementary DNA sequences linkedto the solid support which bind a terminal region of the IRES RNA. Forpurposes of detecting the binding between the IRES RNA and its bindingprotein, the IRES binding-protein can be labeled using any standardmethod known to those skilled in the art including, but not limited to,incorporation of radioactive isotopes or modification by attachment ofligands such as biotin. The latter method enables the practitioner todetect the formation of an immobilized complex using a variety ofcommercially-available biotin detection systems, many of which employ abiotin-binding protein such as streptavidin which in turn traps abiotinylated reporter protein as part of the complex. The reporterprotein may be an enzyme that reacts with a substrate to produce asubstance that can be detected with a spectrophotometer or luminometer.In practice, the test compound and labeled binding protein are incubatedwith the immobilized IRES RNA, on the solid. support, solutioncontaining unreacted binding protein is washed from or otherwise removedthe solid support and the support is analyzed for retained bindingprotein. Test compounds which interfere with binding will reduce theamount of labeled binding protein retained on the support.

[0399] In a second embodiment, assays are used that indirectly measurebinding between the IRES element and its binding protein in the presenceand absence of test compound. In one configuration, the assay relies onthe ability of the binding protein to protect the IRES element RNA fromdegradation by incubation with ribonucleases in vitro (“footprint”assay). More specifically, IRES elements labeled with [³²P] at eithertheir 5′ or 3′ ends are incubated in solution with the purified IRESelement binding protein and test compound under conditions where theIRES element and binding protein form a stable complex in the absence ofthe test compound. Enzymes which cleave RNA, such as ribonuclease T1 orS1, are then added to the assay mixture under predetermined conditionsof temperature and concentration so as to normally cleave each RNAmolecule once in a random fashion. The ribonuclease digestion is haltedby quick chilling and the addition of a chaotropic agent such as ureawhich denatures the ribonuclease and strips bound IRES-binding proteinfrom its RNA. The RNase reaction products (i.e., digested IRES RNA) arefractionated by polyacrylamide gel electrophoresis in the presence ofurea. cleavage of any IRES RNA not bound to and therefore unprotected byan IRES-binding protein will result in the appearance of a “ladder” ofRNA fragments on the gel which are visualized by commonly usedradiographic imaging methods. In contrast, the digestion pattern of anIRES element bound to an IRES-binding protein resembles a ladder withmissing rungs. Potential antiviral compounds which block the interactionbetween the IRES element and its binding protein(s) will restore theladder-like appearance to the digestion profile.

[0400] A second configuration of the indirect binding assay relies onthe well-known ability of nucleic acid binding proteins to alter the gelelectrophoretic migration of nucleic acid fragments to which they arebound. In this assay, IRES RNA labeled, for example, at the 5′ or 3′ endwith [³²P] is incubated in solution with the IRES-binding protein andtest compound under conditions which allow formation of a stable complexbetween the IRES element and its binding protein in the absence of testcompound. The reaction mixture is then fractionated electrophoreticallyon a polyacrylamide gel and the position of the IRES element isvisualized using routine radiographic imaging methods. An IRES elementcomplexed with its binding protein usually migrates more slowly than anunbound IRES element because of the retarding influence of the bulkybinding protein (although in some cases the complex migrates morequickly, presumably because of charge or conformation effects).Potential antiviral compounds which block the interaction between theIRES element and its binding protein(s) will confer a normal rate ofmigration to the IRES element. Any of the above means can be used toidentify compounds in vitro which block the interaction between an IRESelement and its binding protein(s), such interaction being required forIRES element translational activity both in vitro and in vivo. Compoundsidentified in this manner can be further screened as viral translationinhibitors in cell-free translation and whole cell assay systems.

[0401] II. Cell-Free Translation System Assays

[0402] Developing assays to screen for compounds that block IRES elementactivity requires constructing a mRNA molecule characterized by thepresence of a reporter gene the translation of which is under IRESelement control. The level of reporter gene translation is used tomonitor the effect of test compounds on the activity of the controllingIRES element. Preferably, however, the diagnostic mRNA contains not onebut two different reporter genes, wherein the translation of onereporter is under IRES control and translation of the other reporter isunder the control of the host-cell cap structure (m⁷GpppG) and cellular5′-UTR sequence. Such a dicistronic construct makes it possible to usetranslation-based assays to identify compounds that block IRES elementactivity but do not adversely affect the process that cells use toinitiate translation of their own mRNA. In other words, this form of theinvention enables the practitioner to identify compounds that have thedesired mechanism of action while simultaneously eliminating nonspecificand possibly toxic compounds.

[0403] The reporter genes employed for either the monocistronic ordicistronic configurations can be any genes that encode products thatcan be conveniently and reliably detected. Commonly used detectionmethods include, but are not limited to, incorporation of radioisotopes,chemiluminescence, bioluminescence, calorimetric techniques andimmunological procedures. Examples of appropriate reporter genes includeluciferase, chloramphenicol acetyl transferase, secreted embryonicalkaline phosphatase, β-galactosidase, and dihyrodofolate reductase.This list is merely illustrative and in no way limits the scope of theinvention since other suitable reporter genes will be known by thoseordinarily skilled in the art. The method(s) for detecting the reportergene products in the assay are preferably applied directly to thereactions or cells used to screen potential drug activity but, in alesser embodiment, could also be used in conjunction with techniques forfirst fractionating the reaction mixtures. Said techniques, used eithersingly or in combination, may include chromatography, electrophoresis,filtration, ultrafiltration, centrifugation, precipitation, extraction,complex formation or digestion.

[0404] The monocistronic or dicistronic reporter gene constructs can beused for either in vitro or in vivo compound screens. In the in vitro(cell-free) assay format, the desired mRNA construct is encoded by aplasmid DNA molecule which directs transcription of the construct underthe control of a strong promoter, exemplified by the bacteriophage T7 orSP6 promoters. When purified and transcribed in vitro with thehomologous RNA polymerase (e.g. T7 or SP6), the plasmid directs thesynthesis of large amounts of the desired reporter-containing mRNA. Forthe monocistronic assay this mRNA may be transcribed without a capstructure, but for the dicistronic assay, which requires that thetranslation of one of the reporter genes be under the control ofcellular translational zignals, preformed cap structures should bepresent during the transcription to ensure that the mRNA synthesizedcarries a cap-structure at the 5′ end. Either the uncapped monocistronicmRNA or the capped dicistronic mRNA is then used as a template in aeukaryotic in vitro translation system purchased from a commercialsupplier or prepared according to procedures available in the scientificliterature. These mRNAs may be purified prior to use as translationtemplates but, more commonly, purification is not necessary.

[0405] III. Cellular Assays

[0406] Assays that rely on whole cells can be used as primary screens orto screen compounds that pass the in vitro binding assays and cell-freetranslation assays. The cells to be used are first modified eitherstably or transiently (e.g. transfected) with selected reporter geneconstructs. Either the monocistronic or dicistronic construct describedin the preceding section is modified for use in cultured eukaryoticcells by: 1) placing the transcription of the construct under thecontrol of a strong eukaryotic viral promoter, such as SV40, CMV orother promoters commonly used by those skilled in the art, 2) includingsplice signals such as SV40 splice signals to ensure correct processingand transport of RNAs made in the nucleus, and 3) including apolyadenylation signal such as the SV40 signal at the 3′ end of theconstruct so that the reporter mRNA will be synthesized as a 3′polyadenylated molecule.

[0407] A plasmid encoding the construct can be used to establish atransient expression assay for screening compounds that block IRESactivity or, in the preferred embodiment, to establish a stable cellline for screening compounds. The latter may be accomplished byincorporating into the plasmid harboring the desired reporter geneconstruct any of several commonly used selectable markers, such as neo,in order to select and maintain those cells containing the assayplasmid. Alternatively, a stable cell line could be generated byco-transfecting the desired host cells with two plasmids, one containingthe selectable marker and the other containing the dicistronic reportergene construct. Selecting for cells in a co-transfection procedure thathave acquired one plasmid with a selectable marker is a commonly usedway known to those skilled in the art to purify cells which have takenup a second plasmid which lacks the benefit of a selectable marker.

[0408] Also for the stable cell line assay, a reporter gene could bechosen and used, either for the monocistronic or dicistronic construct,that confers a growth advantage to cells exposed to a test compound thatinhibits IRES element activity. More specifically, the reporter geneplaced under IRES element control could be a gene that encodes a productthat inactivates, for example, a drug-resistance pathway in the cell ora pathway that confers resistance to any number of otherwise lethalenvironmental stresses (e.g. temperature, alcohol, heavy metals etc.).Cells containing this reporter gene construct grow poorly or not at allin the presence of the drug or stress, but if the same cells are treatedwith a test compound that inactivates the IRES element activityresponsible for expression of the reporter gene, this gene product willnot be made. Consequently, the pathway under its control will becomeactive and enable the cells to grow in the presence of the environmentalor drug insult.

[0409] The following examples illustrate, but in no way are intended tolimit the present invention.

EXAMPLE 19 Making/Isolating IRES Element RNA Constructs

[0410] A. In Vitro Transcription Reactions

[0411] Oligoribonucleotides are prepared by in vitro transcription fromPCR templates amplified using a 5′ primer containing a T7 promoter byprocedures previously described (Milligan et al., 1987, Nucleic AcidsRes. 15, 8783-8798.). RNAs are labeled by the addition of [a-³²P]-UTP (5μCi) into the transcription reaction. Transcription reactions arepurified using Stratagene NucTrap push columns and eluted with 5 mMHepes pH 7.6, 25 mM KCl, 5 MM MgCl₂ and stored at −20 C.

[0412] B. PCR Reaction

[0413] Amplify selected IRES element from available plasmids usingpolymerase chain reaction (PCR) and primers designed to place T7promoter on 5′ end of PCR fragment. Reaction mixture contains thefollowing: 1 μM primer #1, 1 μM primer #2, 40 μM dATP, 40 μM dGTP, 40 μMdCTP, 40 μM dTTP, 4 pg/μl template DNA, Taq DNA polymerase, 10 mMTris-HCl pH 8.3 25° C., 40 μM KCl, 1.5 mM MgCl₂, and 0.01% (w/v)gelatin.

[0414] The reaction mixture (100 μl total volume) is overlaid with 100μl mineral oil. Dip tube in mineral oil and place in heat block, forcingout air bubbles. Parameters: 94° C. 2 minutes, 42° C. 1 minute, 72° C. 1minute, 2 sec autoextension. Remove as much oil top layer as possible.Add 100 μl TE and extract with CHCl3, then phenol/CHCl3, and finallywith CHCl3. Add 30 μl 3 M NaOAc. Add 600 μl ice-cold EtOH and let standat −20° C. for several hours. Spin 30 minutes at 14K rpm in microfuge,then resuspend in 5 μl H₂O.

[0415] C. Preparation of Internally Labeled IRES RNA for Filter Bindingand UV Cross-Linking Assays

[0416] Reaction mixture contains the following components: 5 μl PCRfragment (5 μl), 0.1% DEPC H₂O (10 μl), 10 mM ATP (5 μl), 10 mM GTP (5μl), 1 mM UTP (2.5 μl), 10 mM CTP (5 μl), [-³²P]-NTP (100 μCi), RNasin(1 μl), and 5×buffer (10 μl; 200 mM Tris pH 8.0 37° C., 50 mM MgCl₂, 25mM DTT, 1 mM spermidine, 40% PEG, 0.5% Triton X-100). The mixture isincubated at 37° C. for 5 minutes, prior to addition of 4 μl T7polymerase (1 mg/ml). The reaction mixture is then incubated at 37° C.for 60 minutes. 2 μl RNase-free DNase is then added, and incubationcontinued at 37° C. for 1 minute. The reaction is then terminated by theaddition of 2 μl 500 mM EDTA and extracted with phenol/CHCl₃. Loadtranscription reaction on column (Stratagene NucTrap push column with 70μl elution buffer (5 mM Hepes pH 7.6, 25 mM KCl, 5 mM MgCl₂). Elute RNAfrom push column with 70 μM elution buffer. Determine cpm/μM withscintillation counter, and store at −20° C. Check integrity of RNA on 6%acrylamide TBE 7M urea gel.

[0417] D. Preparation of End-Labeled IRES RNA for Footprint Assay

[0418] A 500 μM T7 transcription reaction contains: PCR product (50 μl),0.1% DEPC H₂O (320 μl), 100 mM ATP (5 μl), 100 mM GTP (5 μl), 100 mM UTP(5 μl), 100 mM CTP (5 μl), RNasin (5 μl), 5×buffer (100 μl: 200 mM TrispH 8.0 37° C., 50 mM MgCl₂, 25mM DTT, 5mM spermidine, 40% PEG, 0.05%triton X-100), 5′ 37° C., T7 polymerase (1 mg/ml) and 5 μl 60′37° C. 5μl RNase-free DNase, 37° C. 1 minute. Add 10 μl 500 mM EDTA phenol/CHCl₃extract. Wash Stratagene NucTrap and push column with 70 μl elutionbuffer (5 mM Hepes pH 7.6, 25 mM KCl, 5 mM MgCl₂). Load transcriptionreaction on column. Elute RNA from Stratagene NucTrap, push column with70 μl elution buffer. Add H20 to 180 μl and 20 μl 3M NaOAc pH 5.2. Add600 μl ice-cold EtOH, then store at −20° C. overnight. Spin down 14K rpmin microfuge at 4° C.; read A260, then determine concentration. Store at−20° C.

[0419] To 5′-end-label RNA: dephosphorylate cold RNA with calf intestinealkaline phosphatase (0.1 unit/pmol end) in 50 mM NaCl, 10 mM Tris-HClpH 7.9 (25° C.), 10 mM MgCl₂, and 1 mM DTT. Incubate at 37° C. for 60minutes. Extract with phenol/CHCl₃, then CHCl₃ and EtOH precipitate.Phosphorylate RNA with T4 polynucleotide kinase and ³²P-ATP in 70 mMTris-HCl pH 7.6 (25° C.), 10 mM MgCl2, and 5 mM DTT, 37° C. for 30minutes. Extract with phenol/CHCl₃ then CHCl₃ EtOH precipitate, andresuspend in TE. Determine cpm/μl.

[0420] To 3′-end-label RNA: phosphorylate Cp with T4 polynucleotidekinase and ³²P-ATP in 70 mM Tris-HCl pH 7.6 (25° C.), 10 mM MgCl₂, and 5mM DTT, 37° C. for 30 minutes. Ligate ³²P-pCp with cold RNA using T4 RNAligase in 50 mM Tris-HCl pH 7.8 (25° C.), 10 mM MgCl₂, 10 mMmercaptoethanol, and 1 mM ATP, 37° C. 60 minutes. Extract withphenol/CHCl₃ then CHCl₃ EtOH precipitate, and resuspend in TE. Determinecpm/μl.

[0421] E. Construction of pBL and pBCRL Plasmids

[0422] Transcription template pBL was constructed by ligating PCRamplication products of β-globin and luciferase sequences into plasmidvector pUC19. β-globin PCR primers (SEQ. ID NO. 18, SEQ. ID NO. 19) weredesigned to amplify the 5′ non-translated region (“NTR” also referred toas untranslated region, “UTR”) of β-globin and introduce a 5′ EcoR Irestriction site, a 5′ T7 promoter, and a 3′ Kpn I restriction site. TheEcoR I and Kpn I restriction sites were used for ligation into pUC19 togenerate the intermediate plasmid pB. Luciferase PCR primers (SEQ. IDNO. 20, SEQ. ID NO. 21) were designed to amplify the luciferase codingsequence and introduce a 5′ Pst I restriction site and a 3′ Hind IIIrestriction site, for ligation into pB to generate pBL. CAT PCR primers(SEQ. ID NO. 22, SEQ. ID NO. 23) were designed to amplify the CAT codingsequence and introduce a 5′ Kpn I restriction site and a 3. Bam HIrestriction site, for ligation into pBL to generate pBCL. Rhinovirus 145′ NTR PCR primers (SEQ. ID NO. 24, SEQ. ID NO. 25) were designed toamplify the rhinovirus 5′ NTR and introduce a 5′ Bam HI restrictionssite and a 3′ Pst I restriction site which were used to ligate theamplification product into pBCL. Rhinovirus and luciferase start codonsare aligned by transforming the resultant plasmid containing β-globin 5′NTR, CAT, rhinovirus IRES and luciferase sequences into E. coli DMIcells. Unmethylated plasmid DNA is isolated and digested with Bcl I, thedigested plasmid was religated and transformed into E. coli DH5 cells toproduce pBCRL.

[0423] F. Ligation Reaction, Plasmid Screening, and Purification

[0424] DNA fragments were purified on low melting point agarose gels(Maniatis et al., 1989, In: Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.) and ligated with T4 DNA ligase in a 10 μl reactionin 10 mM Tris-HCl pH 7.9 (25° C.), 10 mM MgCl₂ 50 mM NaCl, 1 mM DTT, andincubated overnight at 16° C. Ligated plasmids are transformed into E.coli DH5 or DMI bacterial host cells using rubidium chloride treatment.Transformants harboring plasmid DNA were screened by ampicillinresistance and restriction analysis of minilysate plasmid DNA (Maniatiset al., 1989, In: Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y.). Plasmids were sequenced in the region of interest with T7DNA polymerase using ³⁵S-labeled dATP.

[0425] G. Purification of DNA from LMP Agarose

[0426] Load cut DNA onto 1% LMP agarose gel in TAE with 0.5 ug/ml EtBr.Run gel slowly (25 mA for several hours) for maximum resolution and toavoid melting. Take picture and locate bands to cut out. Quickly cut outband of right size and put in Eppendorf tube. Add 10 μl 1 M Tris-HCl pH8.0, 10 μl 8 M LiCl, bring volume to approximately 200 μl with H₂O. Add200 μl phenol (not phenol/CHCl₃). Melt agarose 70° C. for 5 minutes.Spin 14K rpm 5 minutes (white interphase appears). Remove aqueous phaseand phenol extract again at 70° C. (clear interphase). Extract with 200μl CHCl₃ twice at 25° C. Add 400 μl EtOH and keep −20° C. 1 hour. Spindown, dry pellet, dissolve in 10 μl TE. 10×TAE buffer: 24.2 g TrizmaBase, 5.7 ml glacial acetic acid, 12.5 ml 0.4 M EDTA, bring up to 500 mlwith H₂O.

[0427] H. Ligation

[0428] pBLuc Construction: Ligate 0.1 ug pUC18 (digested with KpnI andSalI) with PCR1 (digested with KpnI and ApaI) and PCR2 (digested withApaI and SalI).

[0429] pBCATIRESLuc Construction: Ligate 0.1 ug pBLuc (digested withXhoI and BclI) with PCR3 (digested with XhoI and NheI) and PCR4(digested with NheII and BclI).

[0430] I. Transformation

[0431] Preparation of Competent Cells: Grow 5 ml of DH5 cells overnight37° C. 2 mls overnight into 100 mls LB in 500 ml flask. Grow to OD=0.48A600 (around 2 hours). Split into two 50 ml fractions and spin in SS34rotor 5 minutes at 4800 rpm, 4° C. Decant supernatants and resuspend byvortexing each fraction in 16 mls Rb1. Combine tubes, then spin in SS34rotor 10 minutes at 4800 rpm, 4° C. Decant supernatant. Gently resuspendcell pellet in 3.2 mls of Rb2 15 minutes 4° C. Quick freeze 200 μlaliquots and store −80° C. for 200 Rb1 MW mls  30 mM KOAC 98.14 589 mg100 mM RbC1₂ 120.9 2.42 g  10 mM CaCl₂—H₂O 147.02 294 mg  50 mMMnCl₂—4H₂O 197.9 1.98 g 15% glycerol 30 mls

[0432] for 200 Rb2 MW mls 10 mM MOPS 209.3 209 mg 10 mM RbCl₂ 120.9 120mg 75 mM CaCl₂—H₂O 147.02 3.1 g 15% glycerol 30 mls

[0433] Transformation: 100 μl competent cells plus DNA. 30 minute 4° C.Heat shock 2 minutes 42° C. Place back on ice, and add 1 ml LB broth(best to transfer to culture tube containing 2 ml LB broth). 37° C. 1 hrwith shaking plate 100 μl on selective plate. Spin down remaining cells,decant, resuspend, and plate on selective plate.

[0434] J. DNA Sequencing with USB Sequenase Kit

[0435] Extract (mini-prep) DNA from 1.5 ml overnight (or 1 ug purifiedDNA). Resuspend in 25 μl TE with RNase A. Put 8 μl of DNA into new tube,and add 2 μl 2M NaOH; 2 mm EDTA 5 minutes 25° C. Add 7 μl primer DNA(2pmol/μl). Add 3 μM 2M NaOAc pH 4.6. Mix gently, then add 75 μl EtOH.45 minutes −80° C. (overnight OK). Spin 15 minutes in microfuge. Drypellet. Dissolve pellet in 8 μl dH₂O, add 9 μl sequence cocktail andincubate 2 minutes 25° C. Dispense 3.5 μl of mixture into four tubes,each containing 2.5 μl ddNTP termination mix. 15 minutes 37° C. Add 4 μlstop solution. Boil 3 minutes. Load 3 μl on 6% acrylamide, 7 M urea gel.Cocktail 2 rxns-far 3 rxns-far 5 rxns-far 5 rxns-close seq buffer 4 6 1010 0.1 M DTT 2 3 5 5 dGTP label 0.8 1.2 2 10 (1/20) mix 35S-dATP 2 3 55.0 H₂O 13 14.4 26 12.0 Sequenase ® 0.5 0.8 1.1 1.1 Mn buffer 5.0

[0436] K. Preparation of Capped RNA for Translation Reactions

[0437] T7 polymerase transcription from plasmid DNA was as follows.

[0438] A 200 μl¹ reaction contains: 5 ug plasmid, 1 mM each NTP, 5 ugcut plasmid DNA (20 μl), 0.1% DEPC H₂O (128 μl), 100 mM ATP (2μl), 10 mMGTP (2 μl), 100 mM UTP (2 μl), 10 mM m⁷GpppG (20 μl), RNasin (1 μl),5×plasmid buffer (40 μl), incubate 5′ 37° C. Add 4 μl polymerase (2-4μl) incubate 60′ 37° C. Add 10 μl RNase-free DNase, incubate 37° C. for1 minute. Add 5 μl 500 mM EDTA. Phenol/CHCl₃ extract. CHCl₃ extract. Add70 μl 0.1% DEPC H₂O. Add 30 μl 3M NaOAc pH 5.2. 900 μl EtOH. −20° C.overnight or −80° C. 30 minutes, resuspend in 25 μl TE. Read A₂₆₀Transcription Buffer: 200 mM Tris pH 8.0 at 37° C., 50 mM MgCl₂, 25 mMDTT, 5 mM spermidine, 250 ug/ml BSA, 0.1% DEPC H₂O (650 μl) , 1M Tris(200 μl), (pH 8.0 @ 37° C., pH 8.4 @ 25° C.), 1M DTT (25 μl), 100 mMspermidine (50 μl), 10 ug/μl BSA (25 μl), 1M MgCl₂ (50 μl), store −20°C. (1000 μl).

[0439] L. construction of Mono- and Dicistronic Plasmids forTransfection Assays

[0440] A dicistronic plasmid (pCMV-Luc-IRES-SEAP) is used to transfectcells and assay for translation in vivo in the presence and absense oftest compounds. pCMV-Luc-IRES-SEAP contains, in order, the SV40replication origin, cytomegalovirus (CMV) promoter, luciferase reportergene, selected IRES element, secreted alkaline phosphatase (SEAP)reporter gene, SV40 splice sites, and SV40 polyA signal. TwopUC118-based constructs (pB-SEAP and pB-Luc-IRES-SEAP) are used toconstruct pCMV-Luc-IRES-SEAP. pB-SEAP contains, in order, a T7polymerase promoter, β-globin 5′ nontranslated region, and SEAP reportergene. pB-Luc-IRES-SEAP is constructed from pB-SEAP and contains, inorder, a T7 polymerase promoter, βglobin 5′ nontranslated region,luciferase reporter gene, selected IRES element, and SEAP reporter gene.Construction of pB-SEAP and pB-Luc-IRES-SEAP is performed by PCRamplification of β-globin 5′ NTR, luciferase coding sequence, IRESelement, and SEAP coding sequence from available plasmids using primerscontaining unique 5′ restriction sites. PCR products containing theβ-globin 5′NTR and SEAP coding region are restriction digested andinserted into pUC118 to produce the monocistroic construct pB-SEAP. Thedicistronic plasmid pB-Luc-IRES-SEAP is created by ligating therestriction digested monocistronic plasmid and restriction digested PCRproducts containing the selected IRES element and luciferase codingregion. The dicistronic plasmid used to transfect cells(pCMV-Luc-IRES-SEAP) is constructed by ligating a blunt-ended Kpn I andApa I fragment containing the LUC-IRES-SEAP coding region ofpB-LUC-IRES-SEAP and Eco RV-digested plasmid vector pcDNAI-neo(Invitrogen) containing cytomegalovirus (CMV) promoter, containing SV40replication origin, splice sites, and polyA signal.

[0441] M. PCR

[0442] Amplify T7 promoter, β-globin 5′ NTR, luciferase reporter gene,IRES element, and SEAP reporter gene using polymerase chain reaction(PCR) described above and primers shown below. PCR 3′ Product 5′ PrimerPrimer Sequence 1 GW2 GW3 T7-β-globin 5′ NTR 5 GW10 GW11 SEAP 4 GW8 GW9IRES element 6 GW22 GW13 luciferase

EXAMPLE 20 Filter Binding Assays for IRES-Binding Proteins

[0443] Polypyrimidine tract binding protein (pPTB, p57; Jang and Wimmer,1990, Genes Dev. 4, 1560-1572; Pestova et al., 1991, J. Virol. 65,6194-6204.; Luz and Beck, 1991, J. Virol. 65, 6486-6494.; Borovjagin etal., 1990, FEBS Lett. 2, 237-240.), La (p52), eIF2/2B (Scheper et al.,1991, Biochem. Biophys. Acta 1089, 220-226.), and p70 and p100 have beenidentified as IRES binding proteins. Filter binding assays for pPTB havebeen established and are described below. Filter binding conditions forthe other purified proteins must be determined. IRES elements targetedinclude those from rhinovirus, coxsackievirus, poliovirus, echovirus,hepatitis A virus, hepatitis B virus, hepatitis C virus, mengo virus,encephalomycarditis virus, foot and mouth disease virus, theiler'smurine encephalomyelitis virus, infectious bronchitis virus, vesicularstomatitis virus, and sendai virus.

[0444] Polypyrimidine Tract Binding Protein (pPTB) is purified from E.coli as a recombinant product which contains 12 amino acids from theexpression vector fused to the pPTB amino terminus. Protein-excessfilter binding assays are performed as follows: typical 25 μl reactionscontain ³²P-internally labeled IRES element, pPTB, and MMK buffer (50 mMMES, pH 5.5, 10 mM KCl, 5 mM MgOAc) and are incubated at 25° C. for10-30 min before filtration in the presence or absense of test compound.Reactions are filtered through Schleicher and Schuell nitrocellulosefilters (0.45 μm pore size) presoaked in MMK buffer. The filters arethen washed with 200 μl of MMK buffer, dried in scintillation vials for20 min at 190° C., and counted in Econolume. All RNAs are heated to 95°C. for 3 min and quick cooled on ice just before use. Backgroundsobtained in the absence of protein are less than 5% of the inputradioactivity and subtracted in all cases. Filtration assays contain³²P-labeled RNA (˜10 pM) and pPTB concentrations from 5 nM to 100 nM.Retention efficiencies of the RNA range from 40% to 60%. Equilibriumbinding constants vary less than a factor of two for independentreplicates.

[0445] Establishment of Filter Binding Assays for Other IRES BindingProteins

[0446] Purified La, eIF2/2B, p70, and p97 are incubated with32P-internally labeled IRES elements under various solution conditionswith pH ranges from 4-9, temperature ranges from 4-50° C., monovalentsalt (Li⁺, Na⁺, K⁺, R^(b)+) concentrations from 0-500 mM, divalent saltBe⁺⁺, Mg⁺⁺, C⁺⁺, Ba⁺⁺) concentrations from 0-50 mM, with counter anionF—, Cl—, Br—, I—, and OAc—.

EXAMPLE 21 Chemical Methods for Detecting IRES-Binding Proteins

[0447] Footprint Assays

[0448] 5′ or 3′ end labeled RNA is incubated with purified pPTB, La,eIF2a, p70 or p97 protein under conditions which allow binding andnuclease activity. Ribonuclease T1 or S1 is added at a determinedconcentration, temperature, and time to give 1 hit/molecule RNA.Reactions are quenched by adding 7 M urea and quick freezing in dryice-EtOH bath. Digested RNA fragments are separated on a 6% acrylamide,7 M urea slab gel. Digestion in absence of protein produces a ladder ofRNA digestion products; protection of RNA from nuclease by protein isobserved as missing bands in ladder. Test compounds which interfere withinteraction will restore ladder of RNA digestion products.

[0449] Cross-Linking Assays

[0450] Ultra-violet light cross-linking assays were performed asdescribed previously (Jang and Wimmer, 1990, Genes Dev. 4, 1560-1572).³²P-labeled RNAs were incubated with 50 μg of HeLa extract in 30 μl ofcross-link buffer (5 mM Hepes pH 7.6, 25 mM KCl, 5 mM MgCl₂, 3.8%glycerol) containing 1 μg rRNA at 30° C. for 20 minutes. Reactions werecross-linked in a Stratagene cross linker for 40 minutes. RNAs weredigested by incubation with 20 μg RNaseA and 200 units of RNase T1.Cross-linked proteins were separated on 12.5% sodium dodecyl sulfate(SDS) polyacrylamide gels using the buffer system of Laemmli (1970,Nature 227, 680-685.), as modified by Nicklin et al., (1987, Proc. Natl.Acad. Sci. USA 84, 4002-4006.). Gels were electrophoresed at 5-10volts/cm at constant current (70 mA), dried, and autoradiographed. Theintensity of the cross-linking signal was quantitated by scanningdensitometry.

EXAMPLE 22 In vitro Translation Screening Assays

[0451] Test compounds are screened for their ability to inhibit viralIRES-directed protein translation in a cell-free system containing anIRES element-protein coding region-containing construct, the selectedcellular binding protein required for viral translation, and cellulartranslation components (ribosomes, etc.).

[0452] A. In Vitro Translation Assay

[0453] Two pUC118-based constructs (pBL and pBCRL, described above) areused to assay for translation in the presence and absense of testcompounds. pBL contains, in order, a T7 polymerase promoter, β-globin 5′nontranslated region, and luciferase reporter gene. pBCRL contains, inorder, a T7 polymerase promoter, β-globin 5′ nontranslated region, CATreporter gene, IRES element, and luciferase reporter gene. Testcompounds are screened for their ability to inhibit luciferase synthesisdriven by an IRES element using construct pBCRL, but not CAT synthesisdriven by a β-globin 5′NTR using construct pBCRL and not luciferasesynthesis driven by β-globin 5′NTR using construct pBL.

[0454] IRES elements targeted include those from rhinovirus,coxsackievirus, poliovirus, echovirus, hepatitis A virus, hepatitis Bvirus, hepatitis C virus, mengo virus, encephalomycarditis virus, footand mouth disease virus, theiler's murine encephalomyelitis virus,infectious bronchitis virus, vesicular stomatitis virus, and sendaivirus.

[0455] B. Preparation of S10 of Hela 53 for Translation:Materials/Preparations

[0456] Rinse Type B homogenizer with EtOH and DEPC H₂O in hood.Hypotonic Lysis Buffer: 0.119 g Hepes (500 μl 1M), 0.049 g KOAc (250 μl2M), 0.016 g MgOAc (74 μl 1M), DEPC H₂O to 50 mls, adjust pH to 7.4 with1 M KOH. Add 25 μl 1 M DTT in 10 ml Hepes buffer, prepare fresh.Dialysis Buffer: 2.383 g Hepes, 8.833 g KOAc, 1.5 ml 1 M MgOAc, H₂O to 1L (non-DEPC H₂O will suffice). Adjust pH to 7.4 with 1 M KOH, add 25 ml1 M DTT in 10 ml Hepes buffer. Autoclave or filter sterilize and store4° C. Dialysis tubing 12000-14000 cutoff. 2×Load Dye: 125 μl 1 MTris-HCl pH 6.8, 400 μl 10% SDS, 100 μl mercaptoethanol, 375 μl 50%glycerol. Add trace bromophenolblue.

[0457] Obtain 2L HeLa S3 cells that are in log-phase (5×105 cells/ml).Wash cells 3 times with ice-cold PBS: (20 ml PBS (10 ml PBS/L cells) for1st wash, 15 ml PBS/L cells for 2nd wash, and 10 ml PBS/L cells for 3rdwash. Spin 2K rpm 10 minutes. Use 30 ml corex tube and HB4 rotor forthird spin. Resuspend to 1.5×packed cell volume with hypotonic bufferand swell on ice 10′. Hypotonic buffer (RNase free): 10 mM K-HEPES pH7.4 1 M stock, 10 mM KOA 4 M stock, 1.5 mM MgOAc (1 M), stock 2.5 mM DTT(add just before use). Homogenize with 15-45 strokes of type Bhomogenizer. Check cell disruption either visually or by dye exclusionassay after 10, 15, 20, 25 etc. strokes. If cells disrupted will seedebris. Spin 5 minutes 2K rpm (remove nuclei). Take supernate and spin20′ at 10K rpm. Use sterile corex tubes. Dialyze 2 hours against 1 L(100 volumes) dialysis buffer (10 mM K-Hepes, pH 7.5, 90 mM KOAc, 1.5 mMMgOAc, 2.5 mM DTT) to clean and replace buffer. Add 2.5 ml 1 M DTT justbefore use. Freeze at −80° C. overnight, thaw at 25° C. approximately 30minutes, immediately place on ice. Spin 10K rpm for 10 minutes inmicrofuge. Add 200 μl 50% glycerol/800 μl lysate supernatant. Add 7.5 μl(2 mg/ml) micrococcal nuclease and 7.5 μl 100 mM CaCl₂ per 1 ml extract.Incubate 25° C. 15 minutes. Add 15 μl 200 mM EGTA/ml extract. Aliquot150 μl/tube, store −80° C.

[0458] C. Translation Reaction

[0459] 10×Translation Mix: 1 mM ATP, 50 μM GTP, 10 mM creatinephosphate, 24 μg/ml CPK, 18 mM Hepes, 2 mM DTT, 24 μg/ml tRNA, 12 μMamino acid mix, 240 μM spermidine. Aliquot and store at −80° C. Mixturecontains the following: 40 μl 100 mM ATP, 6 μl 40 mM GTP, 40 μl 1 Mcreatine phosphate (store −20° C.), 10 μl 10 mg/ml creatine phosphokinase in Hepes (store −20° C.), 76 μl K-Hepes pH 7.6, 8 μl 1 M DTT(thaw at 37° C.), 10 μl 10 mg/ml calf liver tRNA (Boehringer), 50 μlamino acid mix-methionine, 10 μl 100 mM spermidine, and 250 μl H2O to500 μM.

[0460] Master Mix (Prepare Fresh): Mixture contains: 150 μl micrococcalnuclease treated HeLa extract, 50 μl translation mix, 22 μl 2 M KOAc, 3μl 50 mM MgOAc, 16 μl 20 mM MgCl₂, 25 μl 35S-met (20 μCi/μl), sufficientfor 28 translations, for fewer samples take less.

[0461] Translation: Mixture contains: 8.0 μl master mix, 4.5 μl 1 uM RNAin DEPC H₂O, +/−10 μl test compound, incubate 30° C. 3 hours. Add 40 μl2×load dye, 28 μl H₂O, boil 5 minutes, load 20 μl on 12% gel, fix,enhance, expose to XRP film. Try 1M sodium salicylate 16 g/100 ml toenhance.

[0462] D. Luceriferase Assay

[0463] As described by DeWet et al., (1987, Mol. Cell Biol. 7,725-737.). Prepare 1 mM stock solution of D-Luciferin by adding 2.8 mgluciferin (free acid—keep on ice and dark) to 9.8 ml H₂O, vortex toremove clumps, add 100 μl 1M Na₂HPO4 (gives yellow-green color, someprecipitate maybe) add 100 μl 1M NaH₂PO4-H₂O (solution clears); aliquotand store at −20° C. Prepare stock of luciferase in H₂O at 1-10 mg/ml,aliquot, store −20° C. Commercial luciferase dissolved at <1 mg/ml intricine buffer, DTT, MgSO₄, and 0.1% BSA, aliquot, store −20° C. Storetransfected cells (not lysed) at −20° C. 100 μl lysate aliquot, store 4°C. 2-4 weeks. In vitro translation, store −20° C. To perform assays, use350 μl assay buffer at 25° C., add 10-50 μl cold cell supernatant from100 μl lysate, or 1-10 μl from 20 μl in vitro translation reaction.Inject 100 μl luciferin solution. Assay Buffer (use fresh): 125 μl 100mM ATP, 75 μl 1M MgSO4, 4675 μl sonication buffer (100 mM K₂HPO4[dibasic] pH 7.8, 1 mM DTT).

[0464] E. Cellular Assay

[0465] A dicistronic construct directing synthesis of two differentreporter proteins is transfected into cells; cells are exposed to testcompounds, then are tested for their ability to produce each of thereporter proteins. Production of both reporter proteins is visualized ordetected in the same cell preferably simultaneously or alternativelysequentially. The reporter proteins may be any of luciferase,β-galactosidase, secreted embryonic alkaline phosphatase, CAT,β-glucuronidase or other suitable protein as is known in the art.

[0466] Compounds that selectively inhibit viral translation inhibitproduction of reporter protein 2, but not reporter protein 1; compoundsthat are generally toxic to cells inhibit the synthesis of reporterprotein 1 and possibly reporter protein 2.

EXAMPLE 22a Inhibiting Rhinovirus Translation with Antisense DNAOligonucleotide Inhibitors

[0467] The rhinovirus IRES-dependent translation system is an excellenttarget for antiviral compounds since it is essential for rhinovirusinfection and very different than conventional human cellulartranslation systems. A screening assay for rhinovirus IRES-dependenttranslational inhibitors has been established by Applicant and therhinovirus 14 IRES has been shown to be functional in vitro. Using thisassay system, Applicant has identified antisense deoxyoligonucleotidesthat specifically inhibit rhinovirus IRES-dependent translation.

[0468] A. Rhinovirus Translation

[0469] Translational initiation of rhinovirus mRNA has been shown tooccur by a cap-independent non-scanning mechanism, in which the 40Sribosome locates the correct start codon by binding directly to a regionof the viral 5′ NTR, termed the internal ribosomal entry site (IRES)(Borman and Jackson, 188 Virology 685, 1992). Similar IRES-dependenttranslational initiation mechanisms have been proposed for otherpicornaviruses including poliovirus (Pelletier and Sonenberg, 334 Nature320, 1988, and 63 J. Virol. 441, 1989), EMCV (Jang et al., 62 J. Virol.2636, 1988, and 63 J. Virol. 1651, 1989; Molla et al., 356 Nature 255,1992), FMDV (Kuhn et al., 64 J. Virol. 4625, 1990), HAV (Brown et al.,65 J. Virol. 5828, 1991), and an enveloped plus-strand RNA virus,hepatitis C virus (Tsukiyama-Kohara et al., 66 J. Virol. 1476, 1992).

[0470] Rhinovirus belongs to the picornavirus family. The secondarystructures of several picornavirus IRES elements, as well as thehepatitis C virus IRES element, have been proposed (Pilipenko et al.,168 Virology 201, 1989a, and 17 Nucleic Acids Res. 5701, 1989b;Tsukiyama-Kohara et al., 66 J. Virol. 1476, 1992). On the basis of theirnucleotide sequences and proposed secondary structures, IRES elements ofpicornaviruses can be divided into three groups; group I belonging tothe genera Enterovirus and Rhinovirus, group II belonging to the generaCardiovirus and Aphthovirus, and group III belonging to the genusHepatovirus of the Picornaviridae family (Jackson et al., 15 TrendsBiochem. Sci. 477, 1990). Remarkably, the IRES elements between thethree groups share little sequence or structural homology, and none ofthe IRES elements from the three picornavirus groups resemble the IRESelement of hepatitis C virus. The boundaries of the rhinovirus 2,poliovirus 2, and EMCV IRES elements have been determined by making 5′and 3′ deletions of the IRES elements and assaying for cap-independenttranslation (Borman and Jackson, 188 Virology 685, 1992; Nicholson etal., 65 J. Virol. 5886, 1991; Jang and Wimmer, 4 Genes Dev. 1560, 1990).The boundaries determined indicate that all picornavirus IRES elementsare approximately 400 nucleotides (“nts”) long. Although the boundariesof the rhinovirus 14 IRES have not yet been determined, by extrapolatingfrom the above results, it is likely that the 5′ border is near nt 117and the 3′ border is near nt 577 (FIG. 5).

[0471] Oligopyrimidine tracts have been found near the 3′ border of allpicornavirus IRES elements (FIG. 5, nt 572-580). Closer inspection ofthe various oligopyrimidine tracts revealed the presence of a downstreamAUG triplet (FIG. 5, nt 591-593). This conserved element has been termedthe “Y_(n)X_(m)AUG” motif, with Y_(n) corresponding to a pyrimidinetract of length n, wherein n may vary from 4 to 12 and most preferablyfrom 5-9 nucleotides, and X_(m) corresponding to a random spacersequence of length m, wherein m may vary from 5 to 30 and mostpreferably 10-20 nucleotides (Jang et al., 44 Enzyme 292, 1990). Sitedirected and genetic alterations of the “Y_(n)X_(m)AUG” motif suggestthat the sequence of the pyrimidine tract and AUG sequence are importantfor IRES function, as well as proper spacing between the pyrimidinetract and the AUG (Pelletier et al., 62 J. Virol 4486, 1988; Pestova etal., 65 J. Virol. 6194, 1991; Pilipenko et al., 68 Cell 1, 1992). The“Y_(n)X_(m)AUG” motif has been proposed to unify cap-independenttranslation among picornaviruses and may be involved in 18S ribosomalRNA binding (Jang et al., 44 Enzyme 292, 1990; Pilipenko et al., 68 Cell1, 1992). In rhinoviruses and enteroviruses there is also a conserved 21base sequence found upstream of the “Y_(n)X_(m)AUG” motif. It will beevident to one skilled in the art that in the design of an antisenseoligonucleotide effective in inhibiting translation the oligonucleotidewill be complementary to sequences at least partly within the IRES, andsuch sequences will be attractive targets for antisenseoligonucleotides. The importance of this sequence in IRES-dependenttranslation is unknown.

[0472] The start codon used by the rhinovirus IRES element is locatedapproximately 31 nucleotides downstream of the “Y_(n)X_(m)AUG” motif. Ithas been proposed that for rhinoviruses the ribosome binds the IRESelement and then scans to the authentic start codon of the polyprotein(Jackson et al., 15 Trends Biochem. Sci. 477, 1990; Jang et al., 44Enzyme 292, 1990).

[0473] Several cellular proteins have been observed to bind IRESelements or fragments of IRES elements (Witherell et al., 32Biochemistry 8268, 1993; Borman et al., 74 J. Gen. Virol. 1775, 19.93;Meerovitch and Sonenberg, 4 Seminars Virol. 217, 1993; Witherell andWimmer, J. Virol., in press 1994). For some of these proteins there isalso evidence of a functional role in cap-independent translation (Jangand Wimmer, 4 Genes Dev. 1560, 1990; Borman et al., 74 J. Gen. Virol.1775, 1993; Meerovitch et al., 67 J. Virol. 3798, 1993). Two cellularproteins have been found to act synergistically to stimulatecap-independent translation directed by the rhinovirus IRES element(Borman et al., 74 J. Gen. Virol. 1775, 1993).

[0474] B. In Vitro Translation Assay

[0475] To assay translation that is dependent upon the rhinovirus IRESelement in vitro, the dicistronic mRNA (bCRL) is prepared containing theβ-globin 5′ NTR driving translation of the CAT reporter gene andrhinovirus IRES driving translation of the luciferase reporter gene(FIG. 6A). Translational initiation of the CAT reporter in thedicistronic mRNA will be cap-dependent, whereas translational initiationof the luciferase reporter is dependent on the rhinovirus IRES. Acompound that inhibits luciferase expression, without concomitantinhibition of CAT expression, indicates a selective block ofIRES-dependent translational initiation. A control monocistronic mRNA isprepared (bL) containing the β-globin 5′ NTR driving translation of theluciferase reporter gene (FIG. 6B). bL mRNA is used as a control toscreen out compounds that inhibit luciferase activity by inhibitingtranslational elongation or termination of the luciferase reporter gene,shifting the ribosome out of frame, or directly inhibiting enzymaticactivity of the luciferase gene product. bL and bCRL mRNAs are producedby in vitro transcription from plasmids pBL and pBCRL (not shown) usingT7 RNA polymerase (Milligan et al., 15 Nucleic Acids Res. 8783, 1987).

[0476] There are several different ways to quantitate luciferaseactivity. Translation reactions can be performed in HeLa extract, orother cell lines, as described by Sonenberg and co-workers (Lee andSonenberg, 79 Proc. Natl. Acad. Sci. USA 3447, 1982). Translations areperformed with or without micrococcal nuclease treatment of the extractsunder optimal conditions for rhinovirus IRES-dependent translation. Allcomponents of the reaction, including antisense deoxyoligonucleotides,are added to the translation reaction prior to the mRNA. No artificialannealing conditions for binding the antisense deoxyoligonucleotides andmRNA (i.e., high DNA and RNA concentrations, high salt concentrations,or heating and cooling steps) are required. An enhanced luciferase assaykit (available from Analytical Luminescence Laboratory, Promega, orother companies) is used to quantitate luciferase activity. In thisassay, the translation reaction is performed in a well of the microtiterplate at 30° C. for 3 hrs. Buffer(s) from the enhanced luciferase assaykits are added, the sample mixed, and the light emitted from thereaction quantitated by a luminometer or scintillation counter. Theluciferase signal from translation of mRNA is typically >10,000-foldabove the background signal (-mRNA). As an alternative to a commercialluciferase assay kit, a non-enhanced assay described by DeWet et al(1987) could be used. Luciferase and CAT expression, from in vitrotranslation reactions with HeLa extract, can also be quantitated by a[³⁵S]-methionine incorporation assay. [³⁵S]-Methionine incorporation ismeasured by translating bCRL and bL mRNA in the presence of[³⁵S]-methionine, separating the proteins by SDS-PAGE, and visualizingthe bands by autoradiography.

[0477] A transient transfection assay can also be employed using bCRLmRNA and bL mRNA or pCMV-LUC and pCMV-LUC-IRES-SEAP plasmid DNA. bCRLand bL mRNA or pCMV-LUC and pCMV-LUC-IRES-SEAP plasmid DNA is introducedinto HeLa cells, or other cell lines such as 293 or Jurkat, usinglipofectin (Gibco, Inc.), electroporation, or DEAE dextran methods.Luciferase activity from in vivo translation of bCRL and bL mRNA ismeasured by preparing cell extracts using either the triton X-100 orfreeze/thaw method and quantitating light emission. Alternatively,luciferase assays may be performed by growing transiently transfectedcells in a microtiter plate and using a1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) caged luciferinsubstrate (Yang and Thomason, 15 BioTechniques 848, 1993). DMNPE cagedluciferin is generated in a simple one-tube synthesis and requires nofurther purification. The caged luciferin readily crosses the cellmembrane and is cleaved by endogenous esterases, trapping the luciferinsubstrate in the cell. Light output from the cells is proportional toluciferase expression and is quantitated with the luminometer.

[0478] The rhinovirus 14 IRES of bCRL was shown to be functional in theHeLa extract translation system using a ³⁵S-methionine incorporationassay. Translation of dicistronic bCRL mRNA was compared to translationof a dicistronic mRNA, bCXL, containing a reversed and complementarysequence to the rhinovirus IRES. The translation efficiency ofluciferase from bCRL mRNA (driven by the rhinovirus IRES) is as great asthe translation efficiency of CAT driven by the β-globin 5′ NTR (FIG. 7,compare luciferase translation and CAT translation in lanes 7 and 8).Translation of luciferase from dicistronic bCXL mRNA, containing areversed and complementary IRES, is however barely detectable. As aninternal control, translation of CAT (driven by the b-globin 5′ NTR)from bCXL is equivalent to translation of CAT from bCRL. Like therhinovirus IRES element, the reversed and complementary IRES ispredicted to form a high degree of secondary structure that would makescanning through this region unlikely (Jackson et al., 15 TrendsBiochem. Sci. 477, 1990). Luciferase translation from bCRL is thereforedependent on the presence of the IRES in the correct orientation andcannot be due to RNA degradation or alternative translational initiationmechanisms such as termination-reinitiation, leaky scanning, or ribosomejumping. These results provide strong evidence that the rhinovirus IRESin bCRL is functional.

[0479] C. Antisense Oliqodeoxynucleotide Results

[0480] Applicant has designed antisense deoxyoligonucleotides thattarget the 3′ end of the rhinovirus IRES element and inhibit rhinovirusIRES-dependent translation. This region of the IRES was chosen since itcontains both the “Y_(n)X_(m)AUG” motif and the conserved 21 basesequence described above and shown in FIG. 1. Antisensedeoxyoligonucleotide inhibition of the rhinovirus IRES element wasassayed using the [³⁵S]-methionine incorporation assay (FIG. 7) andluciferase activity assay (FIG. 8). An example of an antisenseoligonucleotide that targets this region is anti-IRES-oligo, whichanneals to nts 518-551 of the rhinovirus 14 IRES. The sequence ofanti-IRES-oligo (SEQ. ID NO. 26) is

[0481] 5′ AGTAGTCGGTCCCGTCCCGGAATTGCGCATTACG 3′

[0482] Translation of monocistronic bLuc mRNA (FIG. 6A) and dicistronicbCRL mRNA (FIG. 6B) in the presence and absence of anti-IRES-oligo wasdetermined. As expected, anti-IRES-oligo did not inhibit luciferasetranslation from bLuc mRNA (FIG. 7, compare luciferase translation inlanes 3-4 to lanes 5-6) or CAT from bCRL (FIG. 7, compare CATtranslation in lanes 7-8 with lanes 9-10). Anti-IRES did howeverdramatically inhibit luciferase translation from bCRL mRNA (FIG. 7,compare luciferase translation in lanes 7-8 with lanes 9-10). Thus,anti-IRES-oligo specifically inhibits rhinovirus IRES-dependenttranslation. In addition, modified nucleic acid or nucleic acid analogsas defined in Example 8a may also be utilized in the method of thisexample.

[0483] Luciferase activity assays were performed to quantitate thetranslational inhibition of luciferase from bL and bCRL mRNAs byanti-IRES-oligo. In agreement with the 35S-methionina incorporationassay results, anti-IRES-oligo did not inhibit luciferase translationfrom bL mRNA (FIG. 8, compare lanes 2 and 3) while it inhibitedluciferase translation from bCRL mRNA approximately 95% (FIG. 8, comparelanes 5 and 6). A control deoxyoligonucleotide (control-oligo, notshown) was synthesized with a reversed and complementary sequence toanti-IRES-oligo. The control deoxyoligonucleotide therefore containsapproximately the same G-C and A-T composition, but cannot anneal nts518-551 of the rhinovirus 14 IRES. Control-oligo had no effect on bL orbCRL mRNA translation (FIG. 8, compare lane 4 with lane 2 and lane 7with lane 5). Anti-IRES-oligo thus appears to specifically inhibittranslation driven by the rhinovirus IRES.

EXAMPLE 23 Reporter Gene Assays CAT Spectrophotometric Assay

[0484] The most convenient technique for quantitating the rate of CMacetylation takes advantage of the generation of a free CoA sulfhydrylgroup coincident with transfer of the acetyl group to CM. Reaction ofthe reduced CoA with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) yieldsthe mixed disulfide of CoA and thionitrobenzoic acid and a molarequivalent of free 5-thio-2-nitrobenzoate (Habeeb). The latter has amolar extinction coefficient of 13,600 at 412 nm. The assay is bestcarried out with a recording spectrophotometer equipped with atemperature-controlled cuvette chamber set at 37° C.

[0485] Reagents: Tris.hydrochloride, 1.0 M, pH 7.8, acetyl-CoA, 5 mM,chloramphenicol (D-threo) 5 mM, 5,5′-Dithiobis-2-nitrobenzoic acid(DTNB). The only reagent solution that must be stored frozen inacetyl-CoA. The reaction mixture is freshly prepared from the individualreagents by dissolving 4 mg of DTNB in 1.0 ml of Tris.HCl buffer, afterwhich 0.2 ml of the acetyl-CoA stock solution is added and the totalvolume is made up to 10 ml. The final concentrations of each componentare as follows: Tris.HCl (100 mM), acetyl-CoA (0.1 mM), and DTNB (0.4mg/ml). After the cuvette (1 cm light path) containing enzyme and thereaction mixture has been allowed to equilibrate with the waterbath, thereaction is started by the addition of CM at a final concentration of0.1 mM. The rate of increase in absorption at 412 mM prior to theaddition of CM is subtracted from the observed rate after the start ofthe reaction, and net change in extinction per minute is divided by 13.6to give the result in micromoles per minute of CM-dependent DTNBreacted. Since the latter is equal to the rate of acetylation and since1 unit of CAT=1 μmole of CM acetylated per minute (37° C.), thecalculation also yields the number of units of enzyme in the cuvette.

[0486] An alternative spectrophotometric method can be used if a highconcentration of competing mercaptans interferes with the DTNB assay.The loss of an acyl group from thioesters such as acetyl-CoA isaccompanied by a decrease in absorption in the ultraviolet. Thedifference in molar extinction coefficients of acetyl-CoA and reducedCoA plus acetate is 4500 at 232 nm. Special care must be taken to removeinterfering ultraviolet absorbing material from the enzyme preparationby gel filtration or dialysis. The contribution of the absorption due toprotein added to the cuvette becomes a more serious obstacle in crudeextracts, especially those with low levels of CAT activity. Apart fromthe inconvenience of measurements in the far ultraviolet region and thefact that the method is intrinsically less sensitive than the DTNBprocedure, the assay of thioester cleavage at 232 nm suffers from beinga difference method. The absolute decreases in absorbance per unit timedue to the presence of CM and low levels of CAT may be impossible toquantitate without recourse to the use of a dual beam recordingspectrophotometer.

[0487] Radioisotopic CAT Assay: In this assay chloramphenicol acetyltransferase (CAT) transfers the ³H-labeled acetyl group from acetyl CoAto chloramphenicol bound beads. The beads are washed and counted todetermine CAT activity. This assay is approximately 2-5× more sensitivethan the spectrophotometric assay and will detect CAT in RRL. Materials:chloramphenicol-caproate-agarose (Sigma #C8899), [³H] acetyl-CoA(Amersham TRK.688; specific activity >3Ci/mmol, 250 uCi/ml), acetyl-CoA(Sigma C0378; 100 mM in 50% DMSO [25 mg in 3.1 ml]), chloramphenicol(Sigma C0378; 100 mM in 50% DMSO), CAT (Sigma C8413),. 10XTBS (50 mMTris.HCl [pH 7.5], 150 mM NaCl), wash buffer: TBS containing 5 mMchloramphenicol and 1% SDS. Protocol: Thoroughly resuspend beads insidebottle and pipet 5 ml into Falcon tube. Rinse pipet with 8.5 mls H₂O andput in tube. Add 1.5 ml 10 XTBS and spin 5 K rpm in Sorvall RC6000rotor. Decant supernatant, refill tube with 1XTBS, respin, and decantsupernatant. Add 1XTBS to 5 ml, and store excess beads at 4° C. To 100μl rinsed beads and 2 μM substrate solution (15 mM cold acetyl CoA, 0.65mM [³H] acetyl CoA), add 2 μl CAT standard (dilutions 1:2 to 1:128 inTBS) or 5 μl translation reaction and incubate 20 minutes at 25° C. Add1.25 ml wash buffer to quench reaction, then spin in centrifuge for 5minutes at 14K rpm. Carefully remove supernatant, leaving some liquid onbeads. Repeat wash two more times, then add 100 μl H₂O and vortex.Immediately add scintillation fluid, cap, vortex upside down (to avoidclump of beads at bottom of tube which won't resuspend properly).Measure radioactivity in liquid scintillation spectrometer.

[0488] SEAP Assay: SEAP levels are determined by two distinct assays.The first assay measures the increase in light absorbance at 405 nmwhich accompanies the hydrolysis of p-nitrophenylphosphate (McComb andBowers, 1972, Clin. Chem. 18, 97-104.). This assay is performedessentially as described in Example 16 above.

[0489] The bioluminescence-based assay for SEAP is performed essentiallyas described -(Miska and Geiger, 1987, J. Clin. Chem. Clin. Biochem. 25,23-30.). Fifty μl of freshly prepared substrate solution (0.1 mMD-luciferin-O-phosphate in LUPO buffer (10 mM diethanolamine, 0.5 mMMgCl₂, 10 mM L-homoarginine pH 9.8) and prewarmed to 37° C. for 5minutes in the dark. To this is added 50 μl of heated, clarified medium,prepared as described above, or a medium sample diluted in LUPO buffer.After a 30-minute incubation at 37° C. in the dark, 100 μl of thereaction mixture are transferred into a tube containing 400 μl ofbioluminescence buffer (30 mM Hepes pH 7.75, 5 mM MgCl₂, 0.66 mM EDTA,0.1 mM DTT, 5 mM ATP) containing 1 μl (10⁴ units) of luciferase. Lightimpulses are measured at 37° C. in a luminometer (Berthold Biolumat,Model 9500T—10-s peak-measuring mode). All the chemicals used for theSEAP assays are obtained from Sigma (St. Louis, Mo.) except forluciferase, which is obtained from Boehringer-Mannheim (Indianapolis,Ind.) and D-luciferin-o-phosphate, which can be obtained fromNovabiochem AG, CH-4448, Laufelfingen, Switzerland.

EXAMPLE 24 Cellular Assays

[0490] A dicistronic construct directing synthesis of two differentreporter proteins is transfected into cells; cells are exposed to testcompounds, then are tested for ability to produce reporter proteins.Production of both reporter proteins is preferably simultaneously orsequentially visualized or detected in same cell (luciferase,β-galactosidase).

[0491] A. Appropriate IRES-Reporter Gene Constructs

[0492] A monocistronic plasmid (pCMV-B-SEAP) and disistronic plasmid(pCMV-Luc-IRES-SEAP) are used to transfect cells and assay fortranslation in vivo in the presence and absence of test compounds.pCMV-B-SEAP contains, in order, the SV40 replication origin,cytomegalovirus (CMV) promoter, β-globin 5′-nontranslated region,secreted alkaline phosphatase (SEAP) reporter gene, SV40 splice sites,and SV40 polyA signal. pCMV-Luc-IRES-SEAP contains, in order, the SV40replication origin, cytomegalovirus (CMV) promoter, β-globin 5′nontranslated region, luciferase reporter gene, selected IRES element,SEAP reporter gene, SV40 splice sites, and SV40 polyA signal.

[0493] Test compounds are screened for their ability to inhibit SEAPsynthesis driven by the IRES element from pB-luc-IRES-SEAP, but notinhibit luciferase synthesis dried by β-globin 5′NTR frompCMV-Luc-IRES-SEAP, but not inhibit luciferase synthesis driven byβ-globin 5′NTR from pCMV-Luc-IRES-SEAP and not inhibit SEAP synthesisdriven by β-globin 5′NTR from pCMV-B-SEAP. This screen selects testcompounds which specifically inhibit translation from IRES elementswithout affecting normal cellular translation (from β-globin 5′NTR) orinhibiting SEAP activity.

[0494] IRES elements targeted include those from rhinovirus,coxsackievirus, poliovirus, echovirus, hepatitis A virus, hepatitis Bvirus, hepatitis C virus, mengo virus, encephalomycarditis virus,toot-and-mouth disease virus, theiler's murine encephalomyelitis virus,infectious bronchitis virus, vesicular stomatitis virus, and sendaivirus.

[0495] B. Transfecting Cells with Dicistronic Plasmid

[0496] To denature DNA, mix DNA with 15 μl 20×HBSS (5.0 g Hepes, 8.0 gNaCl, 0.36 g KCl, 0.125 g Na₂HPO₄—H₂O, 1.0 g dextrose, H₂O to 50 ml),.and bring up to 300 μl 1 with H₂O, add 300 μl 1 mg/ml DAE dextran andincubate 4° C. for 30 minutes. Grow COS1 cells on 6 cm plate to 50-70%confluent (100% confluent=complete), wash cells with 2 ml MEM media(+pen−strep, −serum) added to side of plate, tilt plate to cover cells,aspirate off medium by tipping plate and aspirating from side of plate.Repeat wash two more times. Transfect cells by adding 600 μl denaturedDNA to cells at 25° C. for 30 minutes with gentle rocking. Aspirate offdextran from cells, add 2 ml MEM (+2% fetal calf serum at 37° C.) andincubate 37° C. To assay translation, prepare cell extract using TritonX-100 or freeze/thaw method and assay for SEAP and luciferase activityas described above.

EXAMPLE 25 Animal Model(s) of Picornavirus Infection

[0497] Described below are appropriate animal models which may be usedto test potential drugs further. A model in which the infection is“exposed” such as a dermal, buccal, ocular or vaginal model ispreferred.

[0498] A. Infection in Experimental Animals

[0499] A major characteristic of rhinoviruses is a high degree ofspecies specificity. Chimpanzees have been infected with types 14 and 43and gibbons with types 1A, 2, and 14; no overt illnesses were observedin the infected animals (Dick, 1968 Proc. Soc. Exp. Biol. Med. 127,1079-1081; Pinto and Haff, 1969, Nature 224, 1310-1311). Inoculation ofvervent and rhesus monkeys with M (monkey kidney grown) strains of virusdid not produce infection. Infection was not produced in rabbits, guineapigs, weanling mice, or 1-day-old mice injected with human rhinovirusesby the subcutaneous, intraperitoneal, or intravenous route. Similarly,intracranial injections of monkeys, hamsters, or baby mice did notinduce either infection or disease (Hamparian et al., 1961, Proc Soc.Exp. Biol. Med. 108, 444-453; Jackson and Muldoon, 1973, J. Infect. Dis.127, 328-355; Kisch et al., 1964, Am. J. Hyg. 79, 125-133). Intranasalinoculation of ferrets, hamsters, and newborn mice was also withouteffect. One of the animal rhinoviruses, equine rhinovirus, can infectother species including humans (Plummer, 1963, Arch. Ges. Virusforshchr12 694-700.); a hamster model for use in screening of antiviralcompounds has been developed that utilizes this virus. One of the humanrhinoviruses, type 2, was recently adapted to grow in L cells (195);this virus was then used in a mouse model of rhinovirus infection wherein vitro growth was demonstrated (196).

[0500] The cardioviruses (Columbia SK virus, EMC virus, ME virus, MMvirus, and mengovirus) all belong to a single serotype and are here allconsidered to be strains of EMC virus. They are generally regarded asmurine viruses although their host range includes humans, pigs,elephants, and squirrels among others.

[0501] The Theiler's murine encephalomyelitis viruses (TMEV), alsorepresenting a single serotype, are divided into two groups, typified bystrains called GDVII and TO. the GDVII group causes an acute polio-likedisease in mice. The TO group are less virulent and cause a chronicdemyelinating disease resembling multiple sclerosis and have thus becomeimportant models for study of this and other motor neuron diseases(Lipton and Rozhan, 1986, Bhatt, ed., Viral and Mycoplasma Infection ofLaboratory Rodents, pp. 253-276, Academic Press, Orlando.217).

[0502] Apthoviruses (foot-and-mouth disease viruses) infectcloven-footed animals, especially cattle, goats, pigs, sheep, and,rarely, even humans.

[0503] Some picornaviruses, such as cricket paralysis virus (Tinsley etal., 1984, Intervirology 21, 181-186.) infect insects (Longworth, 1978,Adv. Virus Res. 23, 103-157.; Moore and Tinsley, 1982, Arch. Virol. 72,229-245.; Scotti et al., 1981, Adv. Virus Res. 26, 117-142.).

[0504] B. Experimental Infection, Host Range

[0505] The host range of the enteroviruses varies greatly from one typeto the next and even among strains of the same type. They may readily beinduced, by laboratory manipulation, to yield variants that have hostranges and tissue tropisms different from those of wild strains; thishas led to the development of attenuated poliovaccine strains.

[0506] Polioviruses have a very restricted host range among laboratoryanimals (Bodian, 1959, In: Rivers and Horsfall, eds., Viral andRickettsial Infections of Man, Third ed., pp. 430-473, 479-518,Lippincott, Pa.). Most strains will infect and cause flaccid paralysisonly in monkeys and chimpanzees. Infection is initiated most readily bydirect inoculation into the brain or spinal cord. Chimpanzees andcynomolgus monkeys can also be infected by the oral route; inchimpanzees, the infection thus produced is usually asymptomatic. Theanimals become intestinal carriers of the virus; they also develop aviremia that is quenched by the appearance of antibodies in thecirculating blood. Unusual strains have been transmitted to mice orchick embryos.

[0507] The original criteria for classification as a member-of theechovirus group included the provision that the prototype strains failto produce disease in suckling mice or in monkeys. However, differentstrains can produce variants that exhibit animal pathogenicity. A numberof echoviruses have produced inapparent infections in monkeys, with mildlesions in the CNS (Wenner, 1962, Ann NY Acad. Sci. 101, 398-412.). Inthe chimpanzee, no apparent illness is produced, but infection can bedemonstrated by the presence and persistence of virus in the throat andin the feces and by type-specific antibody responses (Itoh and Melnick,1957, J. Exp. Med. 106, 677-688.). Initially, echoviruses weredistinguished from coxsackieviruses by their failure to producepathological changes in newborn mice; this led to the earlyclassification of these strains as coxsackievirus A23. Conversely,strains of some coxsackievirus types (especially A9) lack mousepathogenicity and thus resemble echoviruses. This variability inbiological properties is the chief reason why new members of the genusare no longer being sub-classified as echoviruses or coxsackievirusesbut are simply called enteroviruses.

[0508] The cardinal feature of coxsackieviruses is their infectivity fornewborn mice (Daldorf and Melnick, 1965, In: Horsfall and Tamm, eds.,Viral and Rickettsial Infections of Man, Fourth ed., pp. 474-512,Lippincott, Philadelphia). Chimpanzees and cynomolgus monkeys can beinfected subclinically; virus appears in the blood and throat for shortperiods and is excreted in the feces for 2-5 weeks. Type A14 producespoliomyelitis-like lesions in adult mice and in monkeys, but in sucklingmice this type produces only myositis. Type A7 strains produce paralysisand severe CNS lesions in monkeys (Dalldorf, 1957, J. Exp. Med. 106,69-76.;,268), and at one time this serotype was considered to be afourth type of polio-virus.

[0509] Group A coxsackieviruses characteristically produce widespreadmyositis in the skeletal muscles of newborn mice, resulting in flaccidparalysis without other observable lesions (Daldorf and Melnick, 1965,In:

[0510] Horsfall and Tamm, eds., Viral and Rickettsial Infections of Man,Fourth ed., pp. 474-512, Lippincott, Philadelphia). In addition to beingable to infect the immature skeletal muscles of newborn mice,coxsackieviruses of the A group also can infect surgically denervatedmuscles of adult mice, whereas mature innervated muscles are relativelyresistant. Leg muscles of adult mice in which quantal release ofacetylcholine had been blocked with botulinum toxin were susceptiblewhen subsequently injected with coxsackievirus A2 (Andrew et al., 1984,Science 223, 714-716.). Since the only known action of the toxin is theeffect on acetylcholine release, the findings suggest that synaptictransmission has a role in preventing the susceptibility of skeletalmuscles to coxsackievirus infection.

[0511] Group B viruses can produce a myositis that is more focal indistribution than that produced by viruses of group A, but they alsogive rise to a necrotizing steatitis involving principally the naturalfetal fat lobules (e.g., intrascapular pads, cervical and cephalicpads). Encephalitis is found at times; the animals die with paralysis ofthe spastic type. Some B strains also produce pancreatitis, myocarditis,endocarditis, and hepatitis in both suckling and adult mice. Thecorticosteroids may enhance the susceptibility of older mice toinfection of the pancreas. Normal adult mice tolerate infections withgroup B coxsackieviruses, but in mice subjected to sustained postweaningundernutrition (marasmus), coxsackievirus B3 produces severe disease,including persistence of infective virus in the heart, spleen, liver,and pancreas. Lymphoid tissues are markedly atrophic in marasmicanimals. Transfer of lymphoid cells from normal mice immunized againstthe virus provides virus-infected marasmic mice with significantprotection against severe sequelae (Woodruff and Woodruff, 1971, Proc.Natl. Acad. Sci. USA 68, 2108-2111). These observations support thehypothesis that lymphocyte-mediated defense mechanisms may play animportant role in normal recovery from primary viral infections (Paque,1981, Infect. Immun. 31, 470-479.; Woodruff, 1980, Am J. Pathol. 101,427-478.205,283). Athymic mice exposed to coxsackievirus B3 develop apersistent infection in which the myocardium is affected in adisseminated, multifocal way. The RNA viral genome can readily bedetected in the myocardium by the use of radioactively labeled clonedcoxsackie B3 cDNA (Kanbdolf et al., 1987, Proc. Natl. Acad. Sci. USA 84,6272-6276).

[0512] C. Experimental Infection in Animals and Host Range

[0513] Attempts to transmit HAV to experimental were generallyunsuccessful until the 1960s. An outbreak of infectious hepatitis amongchimpanzee handlers at a United States Air Force base during 1958-1960(Hills, 1961, Am. J. Hyg. 73, 316-328.; Hills, 1963, Transfusion 3,445-453.) restimulated interest in subhuman primates as possible modelsfor human hepatitis. In 1962, Deinhardt et al. (Dienhardt et al., 1962,Am. J. Hyg. 75, 311-321.) described the development of mild liver enzymeabnormalities and histopathologic changes in about two-thirds of 37chimpanzees inoculated with acute-phase serum or feces. Expectations ofjaundice (which rarely occurs in subhuman primates), as well as theassay of aspartate aminotransferase instead of the more sensitive andspecific aminotransferase, served to minimize the significance of theseresults.

[0514] In 1967, Deinhardt, et al. (J. Exp. Med. 125, 673-688.)successfully transmitted and passaged hepatitis in marmosets by usingacute-phase sera from patients with disease that had the epidemiologiccharacteristics of hepatitis A. Interpretation of the results wasinitially hampered by the presence of a latent marmoset agent (or anagent of non-A, non-B hepatitis) in some Saguinus species that wasreactivated by experimental manipulations, resulting in hepatitis (Parksand Melnick, 1969, J. Infect. Dis. 120, 539-547, 548-559.). Theirresults were subsequently confirmed when coded control sera andacute-phase sera from HAV-infected human volunteers ware correctlyidentified upon inoculation into marmosets (Holmes et al., 1971, J.Infect. Dis. 124, 520-521.; Holmes et al., 1969, Science 165, 816-817.).Further evidence for transmission to marmosets and eventually tochimpanzees soon followed (Dienstag et al., 1975, J. Infect. Dis. 132,532-545.; Lorenz et al., Proc. Soc. Exp. Biol. Med. 135, 348-354.;Lundquist et al., 1974, Proc. Natl. Acad. Sci. USA 71, 4774-4777.;Maynard et al., 1975, J. Infect. Dis. 131, 194-196.; Maynard et al.,1975, Am. J. Med. Sci. 270, 81-85.; Provost et al., 1977, Proc. Soc.Exp. Biol. Med. 155, 283-286.).

[0515] HAV produces disease in humans, chimpanzees (Pan troglodytes)(Dienstag et al., 1975, J. Infect. Dis. 132, 532-545.; Lundquist et al.,1974, Proc. Natl. Acad. Sci. USA 71, 4774-4777.; Maynard et al., 1975,J. Infect. Dis. 131, 194-196.; Maynard et al., 1975, Am. J. Med. Sci.270, 81-85.), owl monkeys (Aotus trivirgatus) (LeDuc et al., 1983,Infect. Immun. 40, 766-772.; Lemon, 1982, J. Med. Virol. 10, 25-36.),stump-tailed monkeys (Macaca speciosa) (Mao et al., 1981, J. Infect.Dis. 144, 55-60.), and several species of South American marmoset(tamarin) monkeys (most notably Saquinus mystax and S. labiatus)(Deinhardt et al., 1967, J. Exp. Med. 125, 673-688.; Holmes et al.,1971, J. Infect. Dis. 124, 520-521.; Holmes et al., 1969, Science 165,816-817; Lorenz et al., Proc. Soc. Exp. Biol. Med. 135, 348-354.;Mascoli et al., 1973, Proc. Soc. Exp. Biol. Med. 142, 276-282.; Provostet al., 1977, Proc. Soc. Exp. Biol. Med. 155, 283-286.; Purcell et al.,1975, Am. J. Med. Sci. 270, 61-71.). Disease in nonhuman primatesresembles that in humans but is usually milder. After infecting theseanimals, HAV or viral antigen can usually be detected in serum, liver,bile, and feces.

[0516] Other primate species are susceptible to infection but do notdevelop disease; this limits their usefulness for laboratory studies ofhuman HAV strains (Burke et al., 1981, Lancet, 2, 928.; Burke et al.,1984, AM J. Trop. Med. Hyg. 33, 940-944,; Eichberg et al., 1980, LabAnim. Sci. 30, 541-543.). Cynomolgus monkeys (Macaca fascicularis) werefound to have been infected with HAV in the wild (Burke and Heisey,1984, Am. J. Trop. Med. Hyg. 33, 940-944.). In the laboratory, hepatitiswas induced in M. fascicularis and M. arctoides by experimentalinoculation with the YaM-55 strain of HAV isolated from cynomolgusmonkeys but not by human HAV strain HAS15 (Andzhaparidze et al., 1987,Vopr Virus 2, 440-448.). These data, along with the demonstration ofgenomic differences between the PA21 strain of the HAV isolated from owlmonkeys and the human HAV strain HM175, suggest that host range variantsof HAV may have been selected in subhuman primates (Lemon et al., 1987,J. Virol. 61, 735-742.). In addition, it appears that a host rangealteration can be experimentally induced. After 20 passages inmarmosets, HAV strain MS-1 was more virulent for marmosets but wasattenuated for chimpanzees (Bradley et al., 1984, J. Med. Virol. 14,373-386.).

[0517] Administration of Agents

[0518] In practicing the methods of the invention, the compositions canbe used alone or in combination with one another, or in combination withother therapeutic or diagnostic agents. These compositions can beutilized in vivo, ordinarily in a mammal, preferably in a human, or invitro. In employing them in vivo, the compositions can be administeredto the mammal in a variety of ways, including parenterally,intravenously, subcutaneously, intramuscularly, colonically, rectally,vaginally, nasally, orally, transdermally, topically, ocularly,intraperitoneally, or as suitably formulated surgical implants employinga variety of dosage forms. As will be readily apparent to one skilled inthe art, the useful in vivo dosage to be administered and the particularmode of administration will vary depending upon the mammalian speciestreated, the particular composition employed, and the specific use forwhich these compositions are employed. The determination of effectivedosage levels, that is the dosage levels necessary to achieve thedesired result, will be within the ambit of one skilled in the art.Typically, applications of compositions are commenced at lower dosagelevels, with dosage level being increased until the desired effect isachieved.

[0519] The dosage for the compositions of the present invention canrange broadly depending upon the desired affects and the therapeuticindication. Typically, dosages will be between about 0.01 μg and 100mg/kg, preferably between about 0.01 and 10 mg/kg, body weight.Administration is preferably per os on a daily or as-needed basis.

[0520] Orally-administered formulations can be prepared in conventionalforms, including capsules, chewable tablets, enteric-coated tablets,syrups, emulsions, suspensions, or as solid forms suitable for solutionor suspension in liquid prior to administration. Suitable excipientsare, for example, water, saline, dextrose, mannitol, lactose, lecithin,albumin, sodium glutamate, cysteine hydrochloride or the like. Inaddition, if desired, the pharmaceutical compositions may contain minoramounts of nontoxic auxiliary substances, such as wetting agents, pHbuffering agents, and the like. If desired, absorption enhancingpreparations (e.g., liposomes) may be utilized.

[0521] In selected cases, drug delivery vehicles may be employed forsystemic or topical administration. They can be designed to serve as aslow release reservoir, or to deliver their contents directly to thetarget cell. An advantage of using direct delivery drug vehicles is thatmultiple molecules are delivered per vehicle uptake event. Such vehicleshave been shown to also increase the circulation half-life of drugswhich would otherwise be rapidly cleared from the blood stream. Someexamples of such specialized drug delivery vehicles which fall into thiscategory are liposomes, hydrogels, cyclodextrins, biodegradable polymers(surgical implants or nanocapsules), and bioadhesive microspheres.

[0522] For example, a liposome delivery vehicle originally designed as aresearch tool, Lipofectin, has been shown to deliver intact molecules tocells. Liposomes offer several advantages: They are non-toxic andbiodegradable in composition; they display long circulation half-lives;and recognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost-effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

[0523] Other controlled release drug delivery systems, such asnanoparticles and hydrogels may be potential delivery vehicles for anagent. These carriers have been developed for chemotherapeutic agents.

[0524] Topical administration of agents is advantageous since it allowslocalized concentration at the site of administration with minimalsystemic adsorption. This simplifies the delivery strategy of the agentto the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be administeredis far less than that required for other administration routes.

[0525] Effective delivery requires the agent to diffuse into theinfected cells. Chemical modification of the agent may be all that isrequired for penetration. However, in the event that such modificationis insufficient, the modified agent can be co-formulated withpermeability enhancers, such as Azone or oleic acid, in a liposome. Theliposomes can either represent a slow release presentation vehicle inwhich the modified agent and permeability enhancer transfer from theliposome into the infected cell, or the liposome phospholipids canparticipate directly with the modified agent and permeability enhancerin facilitating cellular delivery.

[0526] Agents may also be systemically administered. Systemic absorptionrefers to the accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes whichlead to systemic absorption include: oral, intravenous, subcutaneous,intraperitoneal, intranasal, intrathecal and ocular. Each of theseadministration routes exposes the agent to an accessible diseasedtissue. Subcutaneous administration drains into a localized lymph nodewhich proceeds through the lymphatic network into the circulation. Therate of entry into the circulation has been shown to be a function ofmolecular weight or size. The use of a liposome or other drug carriercan localize the agent at the lymph node and participate in the deliveryof the agent to the cell.

[0527] A formulation which can associate agents with the surface oflymphocytes and macrophages is also useful. This will provide enhanceddelivery to, for example, HSV-infected cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of infectedcells.

[0528] Intraperitoneal administration also leads to entry into thecirculation with the molecular weight or size of the agent-deliveryvehicle complex controlling the rate of entry.

[0529] Liposomes injected intravenously show accumulation in the liver,lung and spleen. The composition and size can be adjusted so that thisaccumulation represents 30% to 40% of the injected dose. The rest isleft to circulate in the blood stream for up to 24 hours.

[0530] All publications referenced herein are hereby incorporated byreference herein, including the nucleic acid sequences listed in eachpublication.

[0531] Other embodiments are within the following claims.

1 33 1515 base pairs nucleic acid single linear DNA 1 ATGGTGGCCCCCGGCTCTGT GACCAGCCGG CTGGGCTCGG TGTTCCCTTT CCTGCTGGTC 60 CTGGTGGACCTGCAGTACGA AGGTGCTGAA TGTGGAGTAA ATGCAGATGT TGAGAAGCAT 120 CTGGAATTGGGCAAGAAGCT GCTCGCAGCC GGACAGCTCG CGGATGCGTT ATCTCAGTTT 180 CACGCTGCAGTAGATGGTGA CCCTGATAAC TATATTGCTT ACTATCGGAG AGCTACTGTC 240 TTTTTAGCTATGGGCAAATC AAAAGCAGCA CTTCCTGATT TAACTAAAGT GATTGAATTG 300 AAGATGGATTTCACTGCAGC AAGATTACAG AGAGGTCACT TATTACTCAA ACAAGGAAAA 360 CTTGATGAAGCAGAAGATGA TTTTAAAAAA GTGCTCAAGT CAAATCCAAG TGAAAATGAA 420 GAGAAGGAGGCCCAGTCCCA GCTTGTCAAA TCTGATGAAA TGCAGCGTCT GCGCTCACAA 480 GCACTGGATGCCTTTGAGAG CTCAGATTTT ACTGCTGCTA TAACCTTCCT TGATAAGATT 540 TTAGAGGTTTGTGTTTGGGA TGCAGAACTT CGAGAACTTC GAGCTGAATG TTTTATAAAA 600 GAAGGGGAACCTAGGAAAGC GATAAGTGAC TTAAAAGCTT CATCAAAATT GAAAAACGAT 660 AATACTGAGGCATTTTATAA AATCAGCACA CTCTACTATG AACTAGGAGA CCATGAACTG 720 TCTCTCAGTGAAGTTCGTGA ATGTCTTAAA CTTGACCAGG ATCATAAAAG GTGTTTTGCA 780 CACTATAAACAAGTAAAGAA ACTGAATAAG CTGATTGAGT CAGCTGAAGA GCTCATCAAA 840 GAAGGCAGGTACACAGATGC AATCAGCAAA TATGAATCTG TCATGAAAAC AGAGCCAGGT 900 GTTCATGAATATACAATTCG TTCAAAAGAA AGGATTTGCC ACTGCTTTTC TAAGGATGAG 960 AAGCCTGTTGAAGCTATTCG AGTATGTTCA GAAGTTTTAC AGGTGGAACC TGACAACGTG 1020 AATGCTCTGAAAGACCGAGC AGAGGCCTAT TTAATAGAAG AAATGTATGA TGAAGCTATT 1080 CAGGATTATGAAACTGCTCA GGAACACAAT GAGAATGATC AGCAGATTCG AGAAGGTCTG 1140 GAGAAAGCACAGAGGCTACT GAAACAGTCA CAGAGACGAG ATTATTACAA AATCTTGGGA 1200 GTAAAAAGAAATGCCAAAAA GCAAGAAATC ATTAAAGCAT ACCGAAAATT AGCACTGCAG 1260 TGGCACCCAGACAACTTCCA GAACGAAGAA GAAAAGAAAA AAGCTGAGAA GAAGTTCATT 1320 GACATAGCAGCTGCTAAAGA AGTCCTCTCC GATCCAGAAA TGAGGAAGAA GTTTGATGAC 1380 GGAGAAGACCCCCTGGACGC AGAGAGCCAA CAAGGAGGTG GCGGCAACCC TTTCCACAGG 1440 AGCTGGAACTCATGGCAAGG GTTCAGTCCC TTTAGCTCAG GCGGACCTTT TAGATTTAAA 1500 TTCCACTTCAATTAA 1515 47 base pairs nucleic acid single linear DNA 2 AATAGAATTCTAATACGACT CACTATAGGG ACACTTGCTT TTGACAC 47 27 base pairs nucleic acidsingle linear DNA 3 ATAAGGTACC TCTGTCTGTT TTGGGGG 27 39 base pairsnucleic acid single linear DNA 4 AATACTGCAG TGATCATGGA AGACGCCAAAAACATAAAG 39 36 base pairs nucleic acid single linear DNA 5 AATAAAGCTTGGGCCCTTAC AATTTGGACT TTCCGC 36 37 base pairs nucleic acid single linearDNA 6 AATAGGTACC ATGGAGAAAA AAATCACTGG ATATACC 37 27 base pairs nucleicacid single linear DNA 7 AATAGGATCC TTACGCCCCG CCCTGCC 27 27 base pairsnucleic acid single linear DNA 8 AATAGGATCC TTAAAACAGC GGATGGG 27 48base pairs nucleic acid single linear DNA 9 AAAACTGCAG CATGCTGATCACAGTATATG TATATATATG CTGTGACC 48 34 base pairs nucleic acid singlelinear DNA 10 AGTAGTCGGT CCCGTCCCGG AATTGCGCAT TACG 34 20 amino acidsamino acid unknown unknown peptide 11 Ala Glu Ala Tyr Leu Ile Glu GluMet Tyr Asp Glu Ala Ile Gly Asp 1 5 10 15 Tyr Glu Thr Ala 20 25 basepairs nucleic acid single linear DNA 12 GAAGGAAGAT GTATCGATCG AAAGC 2521 base pairs nucleic acid single linear DNA Modified Base 3 WHERE NREPRESENTS INOSINE 13 GCNGTTCTCA GTAAGTCTCT G 21 6 amino acids aminoacid single linear peptide 14 Gln Asp Tyr Glu Thr Ala 1 5 36 base pairsnucleic acid single linear DNA 15 GACTCGAGGA TCCGAATTCT TTTTTTTTTTTTTTTT 36 19 base pairs nucleic acid single linear DNA 16 GACGCGACCATCCGAATTC 19 19 base pairs nucleic acid single linear DNA 17 GCTGAAGAGCTCATCAAAG 19 45 base pairs nucleic acid single linear RNA 18 AGCAAAAGCAGGGUAGAUAA UCACUCACUG AGUGACAUCA AAAUC 45 159 base pairs nucleic acidsingle linear RNA 19 GGGCACUCUU CCGUGGUCUG GUGGAUAAAU UCGCAAGGGUAUCAUGGCGG ACGACCGGGG 60 UUCGAACCCC GGAUCCGGCC GUCCGCCGUG AUCCAUGCGGUUACCGCCCG CGUGUCGAAC 120 CCAGGUGUGC GACGUCAGAC AACGGGGGAG CGCUCCUUU 159159 base pairs nucleic acid single linear RNA 20 GGGCACUCUU CCGUGGUCUGGUGGAUAAAU UCGCAAGGGU AUCAUGGCGG ACGACCGGGG 60 UUCGAACCCC GGAUCCGGCCGUCCGCCGUG AUCCAUGCGG UUACCGCCCG CGUGUCGAAC 120 CCAGGUGUGC GACGUCAGACAACGGGGGAG CGCUCCUUU 159 15 base pairs nucleic acid single linear DNA 21CACCTGGGTT CGACA 15 15 base pairs nucleic acid single linear RNA 22GUGGACCCAA GCUGU 15 15 base pairs nucleic acid single linear DNA 23CGGTAACCGC ATGGA 15 15 base pairs nucleic acid single linear RNA 24GCCAUUGGCG UACCU 15 15 base pairs nucleic acid single linear DNA 25AACCCCGGTC GTCCG 15 15 base pairs nucleic acid single linear RNA 26UUGGGGCCAG CAGGC 15 12 base pairs nucleic acid single linear DNA 27CACCTGGGTT CG 12 12 base pairs nucleic acid single linear RNA 28GUGGACCCAA GC 12 27 base pairs nucleic acid single linear DNA 29TCGAACCCCG GTCGTCCGCC ATGATAC 27 27 base pairs nucleic acid singlelinear RNA 30 AGCUUGGGGC CAGCAGGCGG UACUAUG 27 627 base pairs nucleicacid single linear RNA 31 UUAAAACAGC GGAUGGGUAU CCCACCAUUC GACCCAUUGGGUGUAGUACU CUGGUACUAU 60 GUACCUUUGU ACGCCUGUUU CUCCCCAACC ACCCUUCCUUAAAAUUCCCA CCCAUAUGAA 120 ACGUUAGAAG CUUGACAUUA AAGUACAAUA GGAGGCGCCAUAUCCAAUGG UGUCUAUGUA 180 CAAGCACUUC UGUUUCCCCG GAGCAGGUAU AGGCUGUACCCACUGCCAAA AGCCUUAACC 240 GUUAUCCGCC AACCAACUAC GUAACAGUUA GUACCAUCUUGUUCUUGACU GGACGUUCGA 300 UCAGGUGGAU UUCCCCUCCA CUAGUUUGGU CGAUGAGGCUAGGAAUUCCC CACGGGUGAC 360 CGUGUCCUAG CCUCGUGGCG GCCAACAGCU UAUGCUGGGACGCCCUUUUA AGGACAUGGU 420 GUGAAGACUC GCAUGUGCUU GGUUGUGAGU CUCCGGCCCCUGAAUGCGGC UAACCUUAAC 480 CCUGGAGCCU UAUGCCACGA UCCAGUGGUU GUAAGGUCGUAAUGCGCAAU UCCGGGACGG 540 GACCGACUAC UUUGGGUGUC CGUGUUUCUC AUUUUUCUUCAUAUUGUCUU AUGGUCACAG 600 CAUAUAUAUA CAUAUACUGU GAUCAUG 627 54 basepairs nucleic acid single linear DNA 32 GCGTCGACTA ATACGACTCA CTATAGGGAGTCTTATATAA TAGATATACA AAAC 54 29 base pairs nucleic acid single linearDNA 33 GGGAAATTTT TATTGGCGAG TAAACCTGG 29

We claim:
 1. A composition which inhibits the activity of a viralnucleic acid product and wherein said viral nucleic acid productinhibits the function of a cellular component which regulatestranslations, comprising ava 1 and a pharmaceutically acceptable carrierwherein ava 1 is present in an amount effective for inhibiting theactivity of the viral nucleic acid product.
 2. A composition whichinhibits the activity of a viral nucleic acid product wherein said viralnucleic acid product inhibits the function of a cellular component whichregulates translations, comprising ava 9 or ava 15 and apharmaceutically acceptable carrier and where ava 9 or ava 15 is presentin an amount effective for inhibiting the activity of the viral nucleicacid product.
 3. A method of inhibiting translation of a nucleic acidcontaining an IRES wherein said nucleic acid is obtained from a virus,comprising the step of administering to an organism a nucleic acidfragment complementary to at least a portion of said IRES, wherein theability of the nucleic acid fragment to inhibit translation of the viralnucleic acid containing an IRES is detected by: a) contacting thenucleic acid fragment with a reporter gene construct having thefollowing elements operatively linked: a replication origin, a promoter,a reporter gene and said IRES, under the conditions sufficient to allowtranslation of the reporter gene to occur; b) measuring the level of thetranslation product of the reporter gene exposed to the nucleic acidfragment; and c) comparing the amount in (b) to the level of translationproduct synthesized by the reporter gene construct which was not exposedto the nucleic acid fragment, so that nucleic acid fragments whichinhibit translation of nucleic acids containing said IRES areidentified.
 4. The method of claim 3 wherein said virus is picornavirus.5. The method of claim 3 wherein said virus is selected from the groupconsisting of rhinovirus, enterovirus, cardiovirus and aphthovirus. 6.The method of claim 3 wherein said virus is selected from the groupconsisting of hepatitis A, and hepatitis C.
 7. The method of claim 5wherein said rhinovirus is rhinovirus
 14. 8. The method of claim 3wherein said nucleic acid fragment complementary to at least a portionof said IRES is an oligonucleotide comprising a purine tract of about 4to 12 nucleotides.
 9. The method of claim 3 wherein said nucleic acidfragment complementary to at least a portion of said IRES is anoligonucleotide comprising a purine tract of about 5 to 9 nucleotides.10. The method of claim 3 wherein said oligonucleotide further comprisesa CAT nucleotide triplet.
 11. A method of inhibiting translation of anucleic acid transcript containing an IRES wherein said transcript isobtained from a hepatitis B virus comprising the step of administeringto an organism a nucleic acid fragment complementary to at least aportion of said IRES under conditions sufficient to allow binding of thefragment to a portion of the IRES so that translation of the transcriptis inhibited.
 12. A composition comprising a nucleic acid fragmentcomplementary to at least a portion of a rhinovirus IRES and apharmaceutically acceptable carrier wherein said nucleic acid binds toat least a portion of said IRES and is present in an amount effectivefor inhibiting rhinovirus replication.
 13. The composition of claim 12wherein said nucleic acid fragment complementary to at least a portionof said IRES is an oligonucleotide comprising a purine tract of 4 to 12nucleotides.
 14. The composition of claim 12 wherein said nucleic acidfragment complementary to at least a portion of said IRES is anoligonucleotide comprising a purine tract of 5 to 9 nucleotides.
 15. Thecomposition of claim 12 wherein said nucleic acid fragment complementaryto at least a portion of said IRES which contains a YnXmAUG sequence.16. A composition comprising a nucleic acid fragment wherein saidnucleic acid fragment is complementary to at least a portion ofnucleotides from about 518-551 of a rhinovirus 14 IRES and apharmaceutically acceptable carrier wherein the nucleic acid fragmentbinds to at least a portion of said IRES and is present in an amounteffective for inhibiting rhinovirus 14 replication.
 17. A pharmaceuticalcomposition comprising a nucleic acid fragment complementary to at leasta portion of a viral IRES which contains a YnXm AUG sequence, and apharmaceutically acceptable carrier, wherein the nucleic acid fragmentis present in an amount effective for inhibiting viral replication.